Description of the embodiments
Description of the terms
In the present invention, each term has the following meaning unless otherwise indicated.
In the present invention, when the structure concerned has an isomer, any one of the isomers may be used unless otherwise specified. For example, the structure of cis-trans isomer may be cis-structure or trans-structure, the structure of E/Z isomer may be E-structure or Z-structure, or left-handed or right-handed when optical rotation occurs.
In the invention, the definition of the numerical interval includes both the numerical interval marked by a short transversal line (e.g. 1-6) and the numerical interval marked by a wavy line (e.g. 1-6). In the present invention, unless otherwise specified, an integer interval labeled in the form of an interval may represent a group of all integers within the interval, and the interval includes both endpoints. Such as the integer range 1-6, represents the group consisting of 1,2, 3, 4, 5, 6. Numerical ranges in the present invention, including but not limited to integer, non-integer, percent, fractional, unless otherwise specified, all include both endpoints.
In the present invention, the formulae (2-39) to (2-48) refer to formulae (2-39), formula (2-40), formula (2-41), formula (2-42), formula (2-43), formula (2-44), formula (2-45), formula (2-46), formula (2-47) and formula (2-48).
The numerical values referred to herein are generally within a range of + -10% and may be scaled up to + -15% in some cases, but not more than + -20%. Taking a preset numerical value as a base. For example, a mole percentage of steroid lipids in a solution comprising a solvent of about 40% may be considered to include a mole percentage of steroid lipids of 30% -50%.
In the present invention, the terms "comprising," "including," and "containing," and similar referents in the specification and claims are to be construed to cover both the open and inclusive sense of "including but not limited to".
In the present invention, two or more objects are "each independently preferable", and when having a multi-stage preference, they are not required to be all selected from the group of preference groups of the same level, and one may be a wide range of preference, one may be a small range of preference, one may be a maximum range, and the other may be any preference, and may be selected from the group of preference groups of the same level.
In the present invention, when the divalent linking group such as an alkylene group, an arylene group, an amide bond or the like is not particularly limited, either of the two linking terminals may be selected when it is linked to the other group, for example, when an amide bond is used as a divalent linking group between C-CH2CH2 -and-CH2 -D, it may be C-CH2CH2-C(＝O)NH-CH2 -D or C-CH2CH2-NHC(＝O)-CH2 -D.
In the structural formula of the invention, when the terminal group of the connecting group is easily confused with the substituent contained in the connecting group, the method adoptsTo mark the position of the linking group to which other groups are attached, e.g. in the formulaIn the adopted methodTo mark two positions of the divalent linking group to which other groups are attached, the two formulae respectively representing -CH(CH2CH2CH3)2-、-CH2CH2CH(CH3)2-CH2CH2-.
In the present invention, the range of carbon atoms in a group is marked at the subscript position of C by the subscript form, indicating the number of carbon atoms the group has, e.g., C1-12 indicates "having 1 to 12 carbon atoms", and C1-30 indicates "having 1 to 30 carbon atoms". "substituted C1-12 alkyl" refers to compounds in which the hydrogen atom of the C1-12 alkyl group is replaced. "C1-12 substituted alkyl" refers to compounds having 1 to 12 carbon atoms in the resulting compound after the hydrogen atom of the alkyl group has been replaced. For another example, when a group is selected from C1-12 alkylene, it may be selected from any of the alkylene groups having any number of carbon atoms in the range indicated by the subscript, i.e., may be selected from any of C1、C2、C3、C4、C5、C6、C7、C8、C9、C10、C11、C12 alkylene groups. In the present invention, unless otherwise specified, subscripts labeled in the form of intervals each represent any integer which may be selected from the range, including both endpoints.
The hetero atom in the present invention is not particularly limited, and includes, but is not limited to O, S, N, P, si, F, cl, br, I, B and the like.
In the present invention, the heteroatom for substitution is referred to as a "substitution atom", and any group for substitution is referred to as a "substituent".
In the present invention, "substituted" means any group (e.g., aliphatic hydrocarbon, alkyl, OR alkylene) in which at least one hydrogen atom is replaced by a bond to a non-hydrogen atom such as, but not limited to, halogen atoms such as F, cl, br, and I, oxo (= O), hydroxy (-OH), hydrocarbyloxy (-ORd, where Rd is C1-12 alkyl), carboxy (-COOH), amine (-NRcRc) groups, two Rc groups each independently H, C1-12 alkyl), C1-12 alkyl, and cycloalkyl. In some embodiments, the substituent is a C1-12 alkyl group. In other embodiments, the substituent is cycloalkyl. In other embodiments, the substituent is a halo group, such as fluoro. In other embodiments, the substituent is an oxo group. In other embodiments, the substituent is hydroxy. In other embodiments, the substituent is an alkoxy group. In other embodiments, the substituent is a carboxyl group. In other embodiments, the substituent is an amine group.
In the present invention, "carbon chain linking group" refers to a linking group in which all of the main chain atoms are carbon atoms, while the side chain moiety allows a heteroatom or heteroatom-containing group to replace a hydrogen atom of the main chain carbon. When a "backbone atom" is a heteroatom, it is also referred to as a "backbone heteroatom", e.g., A-S-CH2-B、A-O-CH2 -B,(Atomic interval is denoted as 4) is considered to contain backbone heteroatoms. The carbon chain linking group can be divided into an alkylene group and a carbon chain linking group having a heteroatom in a side group, and the carbon chain linking group having a heteroatom in a side group includes, but is not limited to, oxo (=o), thio (=s), amino (linked to a main chain carbon through a carbon-nitrogen double bond), oxaalkyl in the form of an ether bond, thiaalkyl in the form of a thioether bond, azaalkyl in the form of a tertiary amino group, and the like. The "carbon chain linker" backbone is composed entirely of carbon atoms, and the side groups of the carbon chain are allowed to contain heteroatoms. I.e. by methylene or substituted methylene. The substituted methylene group may be substituted with one monovalent substituent, two monovalent substituents or one divalent substituent (e.g., divalent oxygen, e.g., together with the divalent methylene group form a three-membered ring) And (3) substitution. The substituted methylene can be a hydrogen atom substituted (such as-CH (CH3) -), two hydrogen atoms respectively substituted (such as- (CH3)C(OCH3) -), two hydrogen atoms simultaneously substituted (such as carbonyl, thiocarbonyl, -C (=NH) -, -C (=N+H2) -), or a cyclic side group (such asAtomic separation is noted as 1).
In the present invention, the term "NH-" means that both ends of the secondary amine bond and the term "hydrazine" are blocked with alkylene groups, for example, -CH2-NH-CH2 -, whereas the term "C (=O) -NH-is called an amide bond, and is not considered to contain a secondary amine bond.
In the present invention, for a compound, a group or an atom, it is possible to simultaneously be substituted and hybridized, for example, nitrophenyl substituted for a hydrogen atom, and for example, -CH2-CH2-CH2 -replaced by-CH2-S-CH(CH3) -.
In the present invention, "linkage" means that only linkage is performed, and no atom is contained, and when a group is defined as a linkage, it means that the group may not exist.
In the present invention, "each occurrence is independently" means not only that the inside of a different group can be each independently any option in the definition, but also that each occurrence at a different position in the same group can be each independently any option in the definition, for example, -Z-L4 -Z-, each occurrence of which is each independently -(C＝O)-、-O(C＝O)-、-(C＝O)O-、-O(C＝O)O-、-O-、-S-、-C(＝O)S-、-SC(＝O)-、-NRcC(＝O)-、-C(＝O)NRc-、-NRcC(＝O)NRc-、-OC(＝O)NRc-、-NRcC(＝O)O-、-SC(＝O)NRc- and-NRc C (=o) S-, wherein each occurrence of Rc is each independently a hydrogen atom or a C1-12 alkyl ", in a group" -Z-L4 -Z-, two Z groups can be the same or different, and in a group "-NRcC(＝O)NRc -, two Rc groups can be the same or different, each independently a hydrogen atom or a C1-12 alkyl".
"Group" in the present invention contains at least 1 atom, meaning a radical formed by the loss of one or more atoms from a compound. Groups formed after loss of a portion of a group relative to a compound are also referred to as residues. The valence of the group is not particularly limited and may be classified, for example, into a monovalent group, a divalent group, a trivalent group, a tetravalent group, a..the first place, a one hundred place, and the like. Wherein, the group with valence of 2 or more is collectively called a linking group. The linking group may also contain only one atom, such as an oxy group, a thio group.
In the present invention, "hydrocarbon" means a hydrocarbon compound composed of carbon atoms and hydrogen atoms.
In the present invention, hydrocarbons are classified into aliphatic hydrocarbons and aromatic hydrocarbons according to the hydrocarbon group type. Hydrocarbons that do not contain any structure of benzene rings, hydrocarbyl-substituted benzene rings are defined as aliphatic hydrocarbons. Hydrocarbons containing at least one benzene ring or hydrocarbyl-substituted benzene ring are defined as aromatic hydrocarbons. And the aromatic hydrocarbon may contain an aliphatic hydrocarbon structure such as toluene, diphenylmethane, 2, 3-indane, etc.
In the present invention, the hydrocarbon is classified into saturated hydrocarbon and unsaturated hydrocarbon according to the saturation condition. All aromatic hydrocarbons are unsaturated hydrocarbons. Saturated aliphatic hydrocarbons are also known as alkanes. The degree of unsaturation of the unsaturated aliphatic hydrocarbon is not particularly limited. By way of example, including but not limited to, olefins (containing double bonds), alkynes (containing triple bonds), dienes (containing two conjugated double bonds), and the like. When the aliphatic hydrocarbon portion of the aromatic hydrocarbon is saturated, it is also referred to as an aralkyl hydrocarbon, such as toluene.
In the present invention, the structure of the hydrocarbon is not particularly limited, and may be in the form of a straight chain structure having no side group, a branched structure having a side group, a cyclic structure, a tree structure, a comb structure, a hyperbranched structure, or the like. If not specifically defined, it is preferable that the linear structure not containing a side group, the branched structure containing a side group, and the cyclic structure contain a cyclic structure, and correspond to a linear hydrocarbon, a branched hydrocarbon, and a cyclic hydrocarbon, respectively. Hydrocarbons that do not contain cyclic structures are collectively referred to herein as open-chain hydrocarbons, including, but not limited to, straight-chain structures that do not contain pendant groups, branched-chain structures that contain pendant groups. Open chain hydrocarbons belong to the aliphatic hydrocarbons. The linear hydrocarbon may also be a linear aliphatic hydrocarbon. Branched hydrocarbons may also be branched aliphatic hydrocarbons.
In the present invention, the compounds in which a carbon atom at any position in a hydrocarbon is substituted with a heteroatom are collectively referred to as hetero hydrocarbons.
In the present invention, "hydrocarbon group" refers to a residue formed after a hydrocarbon loses at least one hydrogen atom. According to the number of hydrogen atoms lost, it is classified into monovalent hydrocarbon groups (one hydrogen atom is lost), divalent hydrocarbon groups (two hydrogen atoms are lost, also called hydrocarbylene groups), trivalent hydrocarbon groups (three hydrogen atoms are lost), and so on, and when n hydrogen atoms are lost, the valence state of the hydrocarbon group formed is n. Unless otherwise specified, the hydrocarbon groups in the present invention are particularly monovalent hydrocarbon groups.
The source of the hydrocarbon group in the present invention is not particularly limited, and may be derived from, for example, aliphatic hydrocarbon or aromatic hydrocarbon, saturated hydrocarbon or unsaturated hydrocarbon, straight-chain hydrocarbon, branched-chain hydrocarbon or cyclic hydrocarbon, hydrocarbon or hetero hydrocarbon, and the like. From the point of view of saturation, it may originate, for example, from alkanes, alkenes, alkynes, dienes, etc., from alicyclic or aromatic hydrocarbons, mono-or polycyclic hydrocarbons, for example, from alicyclic or aromatic hydrocarbons, for example.
In the present invention, "aliphatic hydrocarbon group" means a residue formed after an aliphatic hydrocarbon loses at least one hydrogen atom. Unless otherwise specified, the aliphatic hydrocarbon group in the present invention is particularly a monovalent aliphatic hydrocarbon group. The aliphatic hydrocarbon group includes a saturated aliphatic hydrocarbon group and an unsaturated aliphatic hydrocarbon group.
In the present invention, "alkyl" refers to a hydrocarbon group formed from an alkane, and unless otherwise specified, refers to a hydrocarbon group formed by removing a hydrogen atom at any position, and may be linear or branched, and may be substituted or unsubstituted. Specifically, for example, propyl refers to any one of n-propyl and isopropyl, and propylene refers to any one of 1, 3-propylene, 1, 2-propylene and isopropyl.
In the present invention, the term "unsaturated hydrocarbon group" means a hydrocarbon group formed by the unsaturated hydrocarbon losing a hydrogen atom. The hydrocarbon group formed by the unsaturated hydrocarbon losing a hydrogen atom on the unsaturated carbon can be classified into alkenyl group, alkynyl group, dienyl group and the like, as exemplified by propenyl group, propynyl group. The hydrocarbon group formed by the unsaturated hydrocarbon losing a hydrogen atom on the saturated carbon is referred to as an alkenyl group, an alkyne, a diene group, or the like, specifically, an allyl group, a propargyl group, or the like, depending on the unsaturated bond.
In the present invention, "alkenyl" or "alkenyl group" means a substituted or unsubstituted straight or branched alkenyl group comprising two or more carbon atoms (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty or more carbon atoms) and at least one carbon-carbon double bond. The designation "C2-15 alkenyl" means a substituted or unsubstituted straight or branched alkenyl group comprising 2 to 15 carbon atoms and at least one carbon-carbon double bond, i.e., the alkenyl group may comprise one, two, three, four or more carbon-carbon double bonds. Unless specifically stated otherwise, alkenyl groups as described herein refer to both unsubstituted and substituted alkenyl groups.
In the present invention, "alkynyl" or "alkynyl group" means an optionally substituted straight or branched chain hydrocarbon comprising two or more carbon atoms (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty or more carbon atoms) and at least one carbon-carbon triple bond. The label "C2-15 alkynyl" means a substituted or unsubstituted straight or branched alkynyl group including 2 to 15 carbon atoms and at least one carbon-carbon triple bond. Alkynyl groups may include one, two, three, four or more carbon-carbon triple bonds. Unless specifically stated otherwise, alkynyl groups described herein refer to both unsubstituted and substituted alkynyl groups.
In the present invention, the aliphatic hydrocarbon derivative is preferably an ether-derivatized aliphatic hydrocarbon, an aliphatic hydrocarbon derivative having 1 to 2 ether linkages, more preferably an aliphatic hydrocarbon derivative having 2 ether linkages.
In the present invention, "molecular weight" characterizes the mass of a compound molecule, and unless otherwise specified, the measure of "molecular weight" is daltons, da.
In the context of the present invention, the term "about" generally means ± 0.5%.
The "stable presence" and "degradable" of a group in the present invention are a pair of relative concepts, and a detailed example of the stable presence and degradable of a group is given in paragraphs [0134] to [0145] of CN 113402405A.
In the present invention, the "hydroxyl protecting group" includes all groups which can be used as protecting groups for general hydroxyl groups. The hydroxyl protecting group is preferably an alkanoyl group (e.g., acetyl, t-butyryl), aralkanoyl group (e.g., benzoyl), benzyl, trityl, trimethylsilyl, t-butyldimethylsilyl, allyl, acetal or ketal group. The removal of acetyl groups is generally carried out under alkaline conditions, most commonly ammonia hydrolysis of NH3/MeOH and methanolysis catalyzed by methanolic anions, palladium catalyzed hydrogenolysis of benzyl groups in neutral solution at room temperature is easy to remove benzyl groups, metal sodium can be used for reduction cleavage in ethanol or liquid ammonia, trityl groups are generally removed by catalytic hydrogenolysis, trimethylsilyl groups are generally removed by using reagents containing fluoride ions (such as tetrabutylammonium fluoride/anhydrous THF and the like), tert-butyldisilyl ether is stable and can bear ester hydrolysis conditions of alcoholic potassium hydroxide and mild reduction conditions (such as Zn/CH3 OH and the like), fluoride ions (such as Bu4N+F-) can be used for removal in tetrahydrofuran solution, and aqueous acetic acid can be used for removal at room temperature.
In the present invention, the "carboxyl protecting group" means a protecting group which can be converted into a carboxyl group by a deprotection reaction of the carboxyl protecting group by hydrolysis. The carboxyl protecting group is preferably an alkyl group (e.g., methyl, ethyl, t-butyl) or an aralkyl group (e.g., benzyl), more preferably t-butyl (tBu), methyl (Me) or ethyl (Et). In the present invention, the "protected carboxyl group" means a group formed by protecting a carboxyl group with a suitable carboxyl protecting group, and preferably methoxycarbonyl, ethoxycarbonyl, t-butoxycarbonyl, and benzyloxycarbonyl. The carboxyl protecting groups can be removed hydrolytically with acid or base catalysis, occasionally with elimination by pyrolysis, for example tert-butyl can be removed under mildly acidic conditions and benzyl can be removed by hydrogenolysis. The reagent for removing the carboxyl protecting group is selected from TFA, H2 O, liOH, naOH, KOH, meOH, etOH and combinations thereof, preferably a combination of TFA and H2 O, a combination of LiOH and MeOH, or a combination of LiOH and EtOH. The protected carboxyl group is deprotected to yield the corresponding free acid, said deprotection being carried out in the presence of a base, said base and said free acid formed by said deprotection forming a pharmaceutically acceptable salt.
In the present invention, the "amino protecting group" includes all groups which can be used as protecting groups for general amino groups, for example, arylC1-6 alkyl, C1-6 alkoxyC1-6 alkyl, C1-6 alkoxycarbonyl, aryloxycarbonyl, C1-6 alkylsulfonyl, arylsulfonyl, silyl and the like. The amino protecting group is preferably Boc t-butoxycarbonyl, moz p-methoxybenzyloxycarbonyl or Fmoc 9-fluorenylmethoxycarbonyl. The reagent for removing the amino protecting group is selected from TFA, H2 O, liOH, meOH, etOH and combinations thereof, preferably a combination of TFA and H2 O, a combination of LiOH and MeOH, or a combination of LiOH and EtOH. The reagent for removing Boc protecting group is TFA or HCl/EA, preferably TFA. The deprotection agent used for Fmoc protecting group removal was a 20% piperidine in N, N-Dimethylformamide (DMF).
In the invention, the term "carboxyl activation" refers to activation treatment of carboxyl by a carboxyl activating agent, and the carboxyl activation can promote better condensation reaction, such as inhibition of racemization impurity generation in condensation reaction, acceleration of reaction speed by catalysis, and the like. A "carboxyl activating group" is a residue of a carboxyl activator. The carboxyl activating agent is one or more of N-hydroxysuccinimide (NHS), 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI), N-hydroxy-5-norbornene-2, 3-dicarboximide (HONb) and N, N-Dicyclohexylcarbodiimide (DCC), preferably NHS/EDCI, NHS/DCC and HONb/DCC, and most preferably NHS/EDCI.
As used herein, "cationic" means that the corresponding structure is permanently, or non-permanently, capable of carrying a positive charge in response to certain conditions (e.g., pH). Thus, cations include both permanent cations and cationizable cations. Permanent cations refer to the corresponding compounds or groups or atoms that are positively charged at any pH of their environment or hydrogen ion activity. Typically, a positive charge is generated by the presence of a quaternary nitrogen atom. When a compound carries a plurality of such positive charges, it may be referred to as a permanent cation. Cationizable means that a compound or group or atom is positively charged at a lower pH and uncharged at the higher pH of its environment. In addition, in non-aqueous environments where pH cannot be measured, cationizable compounds, groups, or atoms are positively charged at high hydrogen ion concentrations and are uncharged at low hydrogen ion concentrations or activities. It is dependent on the respective properties of the cationizable or polycationizable compound, in particular the pKa of the corresponding cationizable group or atom, at which pH or hydrogen ion concentration it is charged or uncharged. In dilute aqueous environments, the fraction of cationizable compounds, groups or atoms bearing a positive charge can be estimated using the so-called hessian bach (Henderson-Hasselbalch) equation, which is well known to those skilled in the art. For example, in some embodiments, if a compound or moiety is cationizable, it is preferably positively charged at a pH of about 1 to 9, preferably 4 to 9, 5 to 8, or even 6 to 8, more preferably at a pH of equal to or lower than 9, equal to or lower than 8, equal to or lower than 7, most preferably at physiological pH (e.g., about 7.3 to 7.4), i.e., under physiological conditions, particularly under physiological salt conditions of cells in vivo. In other embodiments, it is preferred that the cationizable compound or moiety be primarily neutral at physiological pH (e.g., about 7.0-7.4), but become positively charged at lower pH values. In some embodiments, the preferred range of pKa of the cationizable compound or moiety is from about 5 to about 7.
In the present invention, "cationic lipid" means a lipid containing a positive charge or ionizable as a whole. Cationic lipids, in addition to the structural formula (1) of the present invention, include, but are not limited to, N, N-dioleyloxy-N, N-dimethylammonium chloride (DODAC), N, N-distearyl-N, N-dimethylammonium bromide (DDAB), N- (1- (2, 3-dioleyloxy) propyl) -N, N, N-trimethylammonium chloride (DOTAP), N- (1- (2, 3-dioleyloxy) propyl) -N, N, N-trimethylammonium chloride (DOTMA), N, N-dimethyl-2, 3-dioleyloxy-propylamine (DODMA), 3- (didodecylamino) -N1, N1, 4-tridecyl1-piperazineethylamine (KL 10), N1- [2- (didodecylamino) ethyl ] -N1, N4, N4-tridecyl1, 4-piperazineethylamine (KL 22), 14, 25-ditridecyl-15,18,21,24-tetraaza-trioctadecyl (KL 25), 1, 2-dioleyloxy-N, 3-dioleyloxy-propylamine (DODMA), 3- (didodecylamino) -N1, N1, 4-tridecylamino-ethyl ] -N1, N4-tridecylamino-piperazineethylamine (KL 22), N-2, 4-didodecylamine (DLDLN-N-2) Any one of (i) tricycloheptadeca-6,9,28,31-tetraen-19-yl 4- (dimethylamino) butyrate (DLin-MC 3-DMA) and (ii) 2, 2-diiodo-4- (2-dimethylaminoethyl) - [1,3] -dioxolane (DLin-KC 2-DMA), ((4-hydroxybutyl) azadialkyl) bis (hexane-6, 1-diyl) bis (2-hexyldecanoate) (ALC-0315), heptadec-9-yl-8- ((2-hydroxyethyl) (6-oxo-6- ((undecoxy) hexyl) amino) octanoate) (SM 102), and mixtures thereof.
In the present invention, "pegylated lipid" refers to a molecule comprising a lipid moiety and a polyethylene glycol moiety. The polyethylene glycol lipid includes, but is not limited to, polyethylene glycol-1, 2-dimyristate glyceride (PEG-DMG), polyethylene glycol-distearoyl phosphatidylethanolamine (PEG-DSPE), PEG-cholesterol, polyethylene glycol-diacylglycerol (PEG-DAG), polyethylene glycol-dialkoxypropyl (PEG-DAA), specifically polyethylene glycol 500-dipalmitoyl phosphatidylcholine, polyethylene glycol 2000-dipalmitoyl phosphatidylcholine, polyethylene glycol 500-stearoyl phosphatidylethanolamine, polyethylene glycol 2000-distearoyl phosphatidylethanolamine, polyethylene glycol 500-1, 2-oleoyl phosphatidylethanolamine, polyethylene glycol 2000-2, 3-dimyristoyl glycerol (PEG-DMG), and the like, in addition to the structural formula (2) of the present invention.
In the present invention, "neutral lipid" refers to any of a number of lipid materials, preferably phospholipids, that exist in an uncharged or neutral zwitterionic form at a selected pH. Such lipids include, but are not limited to, 1, 2-dioleoyl-sn-glycero-3-phosphorylcholine (DLPC), 1, 2-dimyristoyl-sn-glycero-phosphorylcholine (DMPC), 1, 2-dioleoyl-sn-glycero-3-phosphorylcholine (DOPC), 1, 2-dipalmitoyl-sn-glycero-3-phosphorylcholine (DPPC), 1, 2-distearoyl-sn-glycero-3-phosphorylcholine (DSPC), 1, 2-dioleoyl-sn-glycero-phosphorylcholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphorylcholine (POPC), 1, 2-di-O-octadecenyl-sn-glycero-3-phosphorylcholine (18:DietherPC), 1-oleoyl-2-cholesteryl hemisuccinyl-sn-glycero-3-phosphorylcholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphorylcholine (DSPC), 1, 2-dioleoyl-sn-glycero-3-phosphorylcholine (C48), 1, 2-dioleoyl-2-oleoyl-glycero-sn-3-phosphorylcholine (SDPC), 1, 2-dioleoyl-glycero-3-phosphorylcholine (N-phosphorylcholine (Lyso PC), 1-dioleoyl-2-glycero-3-phosphorylcholine (POPC) 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine, 1, 2-didodecyloyl-sn-glycero-3-phosphoethanolamine, 1, 2-dioleoyl-sn-glycero-3-phospho-rac- (1-glycero-sodium salt (DOPG), dioleoyl phosphatidylserine (DOPS), dipalmitoyl phosphatidylglycerol (DPPG), palmitoyl Phosphatidylethanolamine (PE), distearoyl-phosphatidylethanolamine (DSPE), palmitoyl phosphatidylethanolamine (DSPE), dioleoyl-phosphatidylethanolamine (DPPE), stearoyl-phosphatidylethanolamine (DPPC), stearoyl-phosphatidylethanolamine (SOtidyl-phosphatidylethanolamine, stearoyl-2-phosphatidylethanolamine (PSOtidyl-PE), stearoyl-phosphatidylethanolamine (SOP), stearoyl-Phosphatidylethanolamine (PSE), any one of palmitoyl phosphatidylcholine, lysophosphatidylcholine, and Lysophosphatidylethanolamine (LPE) and a composition thereof. Neutral lipids may be of synthetic or natural origin.
In the present invention, the "steroid lipid" is selected from any one of cholesterol, fecal sterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, lycorine, ursolic acid, alpha-tocopherol, and mixtures thereof
In the present invention, "amino acid residue" includes amino acids in which a hydrogen atom is removed from an amino group and/or a hydroxyl group is removed from a carboxyl group and/or a hydrogen atom is removed from a mercapto group and/or an amino group is protected and/or a carboxyl group is protected and/or a mercapto group is protected. Needless to say, an amino acid residue may be referred to as an amino acid. The source of the amino acid in the present invention is not particularly limited unless otherwise specified, and may be a natural source, a non-natural source, or a mixture of both. The type of amino acid structure in the present invention is not particularly limited unless otherwise specified, and may be either L-form or D-form, or a mixture of both.
The term "source of functional groups" in the present invention refers to reactive or potentially reactive, photosensitive or potentially photosensitive, targeted or potentially targeted. The term "latent" refers to being capable of emitting light or targeting upon external stimuli selected from the group consisting of, but not limited to, functionalization modifications (e.g., grafting, substitution, etc.), deprotection, salt complexation and decomplexing, ionization, protonation, deprotonation, change of leaving groups, etc., to a reactive group. The luminescence is not particularly limited, and includes, but is not limited to, visible light, fluorescence, phosphorescence, and the like.
The modified form in the present invention refers to a structural form that can be converted into a target reactive group through any one of chemical change processes such as oxidation, reduction, hydration, dehydration, electron rearrangement, structural rearrangement, salt complexation and decomplexing, ionization, protonation, deprotonation, substitution, deprotection, change of a leaving group, and the like.
The term "modification of a reactive group" as used herein refers to a form of a reactive group that remains active (remains a reactive group) after at least one chemical change, such as oxidation, reduction, hydration, dehydration, electron rearrangement, structural rearrangement, salt complexation and decomplexing, ionization, protonation, deprotonation, substitution, deprotection, change of leaving group, or an inactive form after protection.
The term "micro-modification" in the present invention refers to a chemical modification process that can be completed through a simple chemical reaction process. The simple chemical reaction process mainly refers to chemical reaction processes such as deprotection, salt complexation and decomplexing, ionization, protonation, deprotonation, leaving group conversion and the like, and the micro-variation corresponds to the micro-modification, and refers to a structural form capable of forming a target reactive group after undergoing the simple chemical reaction processes such as deprotection, salt complexation and decomplexing, ionization, protonation, deprotonation, leaving group conversion and the like. The transition of the leaving group, such as the transition from the ester form to the acid chloride form.
In the present invention, "N/P ratio" refers to the molar ratio of ionizable nitrogen atoms in the cationic lipid to phosphate in the nucleic acid.
In the present invention, "nucleic acid" refers to DNA or RNA or modified forms thereof.
As used herein, "RNA" refers to ribonucleic acid that may be naturally occurring or non-naturally occurring. For example, RNA can include modified and/or non-naturally occurring components, such as one or more nucleobases, nucleosides, nucleotides, or linkers. The RNA can include cap structures, chain terminating nucleosides, stem loops, polyadenylation sequences, and/or polyadenylation signals. The RNA may have a nucleotide sequence encoding a polypeptide of interest. For example, the RNA may be messenger RNA (mRNA). Translation of an mRNA encoding a particular polypeptide, for example, in vivo within a mammalian cell, can result in the encoded polypeptide. The RNA may be selected from the non-limiting group consisting of small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), mRNA, single-stranded guide RNA (sgRNA), cas9 mRNA, and mixtures thereof.
In the present invention, FLuc mRNA is capable of expressing a luciferase protein that emits bioluminescence in the presence of a luciferin substrate, so FLuc is commonly used in mammalian cell culture to measure gene expression and cell activity.
In the present invention, methods for determining the expression level of a target gene include, but are not limited to, dot blotting, northern blotting, in situ hybridization, ELISA, immunoprecipitation, enzyme action, and phenotypic assay.
In the present invention, "transfection" refers to the introduction of a species (e.g., RNA) into a cell. Transfection may occur, for example, in vitro, ex vivo, or in vivo.
In the present invention, an "antigen" typically refers to a substance that can be recognized by the immune system, preferably by the adaptive immune system, and is capable of triggering an antigen-specific immune response, for example by forming antibodies and/or antigen-specific T cells as part of the adaptive immune response. Typically, the antigen may be or may comprise a peptide or protein that may be presented to T cells by MHC. An antigen in the sense of the present invention may be a translation product of a provided nucleic acid molecule, preferably an mRNA as defined herein. Fragments, variants and derivatives of peptides and proteins comprising at least one epitope are also understood in this context as antigens.
In the present invention, "delivery" refers to providing an entity to a target. For example, the drug and/or therapeutic agent and/or prophylactic agent is delivered to a subject that is tissue and/or cells of a human and/or other animal.
By "pharmaceutically acceptable carrier" is meant a diluent, adjuvant, excipient or vehicle with which the therapeutic agent is administered, and which is suitable for contacting the tissues of humans and/or other animals within the scope of sound medical judgment without undue toxicity, irritation, allergic response, or other problem or complication commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable carriers that may be used in the pharmaceutical compositions of the present invention include, but are not limited to, sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. When the pharmaceutical composition is administered intravenously, water is an exemplary carrier. Physiological saline and aqueous solutions of glucose and glycerol can also be used as liquid carriers, in particular for injections. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, maltose, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol and the like. The composition may also contain minor amounts of wetting agents, emulsifying agents, or pH buffering agents, as desired. Oral formulations may contain standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. Specifically, for example, excipients include, but are not limited to, anti-adherents, antioxidants, binders, coatings, compression aids, disintegrants, dyes (pigments), demulcents, emulsifiers, fillers (diluents), film formers or coatings, flavors, fragrances, glidants (flow enhancers), lubricants, preservatives, printing inks, adsorbents, suspending or dispersing agents, sweeteners, and hydration water. More specifically excipients include, but are not limited to, butylated Hydroxytoluene (BHT), calcium carbonate, dicalcium phosphate, calcium stearate, croscarmellose sodium, crospovidone, citric acid, crospovidone, cysteine, ethylcellulose, gelatin, hydroxypropyl cellulose, hydroxypropyl methylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methyl parahydroxybenzoate, microcrystalline cellulose, polyethylene glycol, polyvinylpyrrolidone, povidone, pregelatinized starch, phenyl parahydroxybenzoate, retinol palmitate, shellac, silica, sodium carboxymethylcellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (corn), stearic acid, sucrose, talc, titanium dioxide, vitamin a, vitamin E (alpha-tocopherol), vitamin C, xylitol.
The pharmaceutical compositions of the present invention may act systematically and/or locally. For this purpose, they may be administered by a suitable route, for example by injection (e.g. intravenous, intra-arterial, subcutaneous, intraperitoneal, intramuscular injection, including instillation) or transdermally, or orally, buccally, nasally, transmucosally, topically, in the form of an ophthalmic formulation or by inhalation. For these routes of administration, the pharmaceutical compositions of the present invention may be administered in suitable dosage forms. Such dosage forms include, but are not limited to, tablets, capsules, lozenges, hard candies, powders, sprays, creams, ointments, suppositories, gels, pastes, lotions, ointments, aqueous suspensions, injectable solutions, elixirs, syrups.
In the present invention, a vaccine is a prophylactic or therapeutic material that provides at least one antigen or antigen function. The antigen or antigen function may stimulate the adaptive immune system of the body to provide an adaptive immune response.
The present invention, treatment, refers to the treatment and care of a patient to combat a disease, disorder or condition, and is intended to include delaying the progression of the disease, disorder or condition, alleviating or alleviating symptoms and complications, and/or curing or eliminating the disease, disorder or condition. The patient to be treated is preferably a mammal, in particular a human.
Detailed Description
One embodiment of the invention is as follows:
1.1. A cationic lipid is characterized by having a structure represented by a general formula (1):
Wherein X is N or CRa, and Ra is H or C1-12 alkyl;
L1、L2 is each independently any one of a bond 、-O(C＝O)-、-(C＝O)O-、-O(C＝O)O-、-C(＝O)-、-O-、-O(CRcRc)sO-、-S-、-C(＝O)S-、-SC(＝O)-、-NRcC(＝O)-、-C(＝O)NRc-、-NRcC(＝O)NRc-、-OC(＝O)NRc-、-NRcC(＝O)O-、-SC(＝O)NRc- and-NRc C (=o) S-, wherein Rc is each independently at each occurrence a hydrogen atom or a C1-12 alkyl group and S is 2, 3 or 4;
l3 is a bond or a divalent linking group;
Each B1、B2 is independently a bond or C1-30 alkylene;
r1、R2 are each independentlyC1-30 aliphatic hydrocarbon group or C1-30 aliphatic hydrocarbon derivative residue, and at least one of R1、R2 isWherein t is an integer from 0 to 12, and each Re、Rf is independently any one of C1-C15 alkyl, C2-C15 alkenyl, and C2-C15 alkynyl;
R3 is a hydrogen atom 、-Rd、-ORd、-NRdRd、-SRd、-(C＝O)Rd、-(C＝O)ORd、-O(C＝O)Rd、-O(C＝O)ORd orWherein each occurrence of Rd is independently C1-12 alkyl, two Rd groups in NRdRd may be joined to form a ring, G1 is a terminal branching group of k+1 valence, j is 0 or 1, F contains a functional group R01, when j is 0, G1 is absent, when j is 1, G1 gives k F, and k is an integer from 2 to 8;
The alkyl, alkylene, aliphatic derivative residue, alkenyl, and alkynyl groups are each independently substituted or unsubstituted.
1.1.1.X
In the present invention, each occurrence of X is independently N or CRa, wherein Ra is H or C1-12 alkyl.
1.1.2.L1、L2、L3、L4、L5、L7、L8、Z、Z1、Z2
The structure of ,L1、L2、L3、L4、L5、L7、L8、Z、Z1、Z2 in the present invention is not particularly limited, and each independently includes, but is not limited to, a linear structure, a branched structure, or a cyclic structure.
In the present invention, the number of non-hydrogen atoms of ,L1、L2、L3、L4、L5、L7、L8、Z、Z1、Z2 is not particularly limited, but each is independently preferably 1 to 50 non-hydrogen atoms, more preferably 1 to 20 non-hydrogen atoms, and still more preferably 1 to 10 non-hydrogen atoms. The non-hydrogen atom is a carbon atom or a heteroatom. Such heteroatoms include, but are not limited to O, S, N, P, si, B and the like. When the number of non-hydrogen atoms is 1, the non-hydrogen atoms may be carbon atoms or hetero atoms. The number of non-hydrogen atoms is not particularly limited, and may be 1, 2 or more, and the number of non-hydrogen atoms is more than 1, and may be any one of carbon atoms and carbon atoms, carbon atoms and heteroatoms, and heteroatoms.
In the present invention, two identical or different reactive groups may react to form a divalent linking group. The reaction conditions, which are related to the type of divalent linking group formed by the reaction, may be those of the prior art. For example, amino groups are reacted with reactive esters, formate reactive esters, sulfonate esters, aldehydes, α, β -unsaturated bonds, carboxylic acid groups, epoxides, isocyanates, isothiocyanates to give divalent linking groups such as amide groups, urethane groups, amino groups, imine groups (which may be further reduced to secondary amino groups), amino groups, amide groups, amino alcohols, urea linkages, thiourea linkages, and the like; the sulfhydryl is respectively reacted with active ester, formic acid active ester, sulfonate, sulfhydryl, maleimide, aldehyde, alpha, beta-unsaturated bond, carboxylic acid group, iodoacetamide and anhydride to obtain bivalent connecting groups such as thioester, thiocarbonate, thioether, disulfide, thioether, thiohemiacetal, thioether and imide, the unsaturated bond is reacted with the sulfhydryl to obtain thioether group, carboxyl or acyl halide is respectively reacted with sulfhydryl and amino to obtain groups such as thioester group and amido, hydroxyl is reacted with carboxyl, isocyanate, epoxide and chloroformyloxy to obtain bivalent connecting groups such as ester group, carbamate group, ether bond and carbonate group, carbonyl or aldehyde group is reacted with amino, hydrazine and hydrazide to obtain bivalent connecting groups such as imine bond, hydrazone and hydrazone, and the like, and the reactive groups such as azide, alkynyl, alkenyl, sulfhydryl, azide, diene, maleimide, 1,2, 4-triazoline-3, 5-dione, dithioester, hydroxylamine, hydrazide, acrylic ester, allyloxy, isocyanate and tetrazole can be reacted to generate triazole with various structures including but not limited to generate triazole structures.
L1、L2、L3、L4、L5、L7、L8、Z、Z1、Z2 The stability of any one of the divalent linking groups or any one of the divalent linking groups consisting of adjacent hetero atom groups is not particularly limited, and each is independently a stably existing linking group STAG or a degradable linking group DEGG.
1.1.2.1.L1、L2
In the present invention, L1、L2 is each independently any one of a linkage 、-O(C＝O)-、-(C＝O)O-、-O(C＝O)O-、-C(＝O)-、-O-、-O(CRcRc)sO-、-S-、-C(＝O)S-、-SC(＝O)-、-NRcC(＝O)-、-C(＝O)NRc-、-NRcC(＝O)NRc-、-OC(＝O)NRc-、-NRcC(＝O)O-、-SC(＝O)NRc- and-NRc C (=o) S-, wherein Rc is each independently at each occurrence a hydrogen atom or a C1-12 alkyl group and S is2, 3 or 4.
In a specific embodiment of the present invention, more preferably L1、L2 is one of the following:
In case (1), L1、L2 is one of the bond and the other is either -O(C＝O)-、-(C＝O)O-、-O(C＝O)O-、-C(＝O)-、-O-、-O(CRcRc)sO-、-S-、-C(＝O)S-、-SC(＝O)-、-NRcC(＝O)-、-C(＝O)NRc-、-NRcC(＝O)NRc-、-OC(＝O)NRc-、-NRcC(＝O)O-、-SC(＝O)NRc- or-NRc C (=O) S-;
L1、L2 is a connecting bond;
In case (3), L1、L2 are each independently selected from any one of -O(C＝O)-、-(C＝O)O-、-O(C＝O)O-、-C(＝O)-、-O-、-O(CH2)sO-、-S-、-C(＝O)S-、-SC(＝O)-、-NHC(＝O)-、-C(＝O)NH-、-NHC(＝O)NH-、-OC(＝O)NH-、-NHC(＝O)O-、-SC(＝O)NH- and-NHC (=O) S-.
In a specific embodiment of the present invention, more preferably, each L1、L2 is independently selected from any one of-O (c=o) -, - (c=o) O-, and-O (c=o) O-.
In a specific embodiment of the present invention, more preferably one of L1、L2 is- (c=o) O-, the other is-O (C=O) -or- (C=O) O-.
In a specific embodiment of the present invention, more preferably, L1 and L2 are both- (c=o) O.
In a specific embodiment of the invention Rc is preferably a hydrogen atom, or Rc is preferably C1-12 alkyl, more preferably C1-8 alkyl, more preferably any of methyl, ethyl, propyl, butyl, pentyl, hexyl.
1.1.2.2.L7、L8
In the present invention, L7、L8 is each independently a bond or a divalent linker selected from any one of -O(C＝O)-、-(C＝O)O-、-O(C＝O)O-、-C(＝O)-、-O-、-S-、-C(＝O)S-、-SC(＝O)-、-NRcC(＝O)-、-C(＝O)NRc-、-NRcC(＝O)NRc-、-OC(＝O)NRc-、-NRcC(＝O)O-、-SC(＝O)NRc- and-NRc C (=o) S-, each occurrence of Rc is independently a hydrogen atom or a C1-12 alkyl group.
In a specific embodiment of the invention Rc is preferably a hydrogen atom, or Rc is preferably C1-12 alkyl, more preferably C1-8 alkyl, more preferably any of methyl, ethyl, propyl, butyl, pentyl, hexyl.
1.1.2.3.L3
In the present invention, L3 is a bond or a divalent linking group.
In a specific embodiment of the invention, L3 is a divalent linking group, preferably a divalent linking group formed by any one, any two or any combination of two or more of L4、L5 and Z divalent linking groups, more preferably any one of-L4-、-Z-L4-Z-、-L4-Z-L5-、-Z-L4-Z-L5 -and-L4-Z-L5 -Z-, wherein L4、L5 is a carbon chain linking group, each independently is -(CRaRb)t-(CRaRb)o-(CRaRb)p-,t、o、p is an integer of 0-12, each t, O and p is not simultaneously 0, each Ra and Rb is independently a hydrogen atom or a C1-12 alkyl group, each Z is independently at each occurrence -(C＝O)-、-O(C＝O)-、-(C＝O)O-、-O(C＝O)O-、-O-、-S-、-C(＝O)S-、-SC(＝O)-、-NRcC(＝O)-、-C(＝O)NRc-、-NRcC(＝O)NRc-、-OC(＝O)NRc-、-NRcC(＝O)O-、-SC(＝O)NRc- and-NRc C (=O) S-, wherein each Rc is independently H or C1-12 alkyl, and each C1-12 alkyl is substituted or unsubstituted, preferably any one of methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl and octyl.
In one embodiment of the present invention, Rc in the above L3 is preferably a hydrogen atom.
In a specific embodiment of the present invention, L3 is more preferably any one of -(CH2)t-、-(CH2)tZ-、-Z(CH2)t-、-(CH2)tZ(CH2)t- and-Z (CH2)t Z-, where t is an integer from 1 to 12, Z is any one of -(C＝O)-、-O(C＝O)-、-(C＝O)O-、-O(C＝O)O-、-O-、-S-、-C(＝O)S-、-SC(＝O)-、-NRcC(＝O)-、-C(＝O)NRc-、-NRcC(＝O)NRc-、-OC(＝O)NRc-、-NRcC(＝O)O-、-SC(＝O)NRc- and-NRc C (=O) S-, more preferably L3 is any one of -(CH2)t-、-(CH2)tO-、-(CH2)tC(＝O)-、-(CH2)tC(＝O)O-、-(CH2)tOC(＝O)-、-(CH2)tC(＝O)NH-、-(CH2)tNHC(＝O)-、-(CH2)tOC(＝O)O-、-(CH2)tNHC(＝O)O-、-(CH2)tOC(＝O)NH-、-(CH2)tNHC(＝O)NH-、-O(CH2)t-、-C(＝O)(CH2)t-、-C(＝O)O(CH2)t-、-OC(＝O)(CH2)t-、-C(＝O)NH(CH2)t-、-NHC(＝O)(CH2)t-、-OC(＝O)O(CH2)t-、-NHC(＝O)O(CH2)t-、-OC(＝O)NH(CH2)t-、-NHC(＝O)NH(CH2)t-、-(CH2)tO(CH2)t-、-(CH2)tC(＝O)(CH2)t-、-(CH2)tC(＝O)O(CH2)t-、-(CH2)tOC(＝O)(CH2)t-、-(CH2)tC(＝O)NH(CH2)t-、-(CH2)tNHC(＝O)(CH2)t-、-(CH2)tOC(＝O)O(CH2)t-、-(CH2)tNHC(＝O)O(CH2)t-、-(CH2)tOC(＝O)NH(CH2)t-、-(CH2)tNHC(＝O)NH(CH2)t-、-O(CH2)tO-、-C(＝O)(CH2)tC(＝O)-、-C(＝O)O(CH2)tC(＝O)O-、-OC(＝O)(CH2)tOC(＝O)-、-C(＝O)O(CH2)tOC(＝O)-、-OC(＝O)(CH2)tC(＝O)O-、-OC(＝O)O(CH2)tOC(＝O)O-、-C(＝O)NH(CH2)tC(＝O)NH-、-NHC(＝O)(CH2)tNHC(＝O)-、-NHC(＝O)(CH2)tC(＝O)NH-、-C(＝O)NH(CH2)tNHC(＝O)-、-NHC(＝O)O(CH2)tNHC(＝O)O-、-OC(＝O)NH(CH2)tOC(＝O)NH-、-NHC(＝O)O(CH2)tOC(＝O)NH-、-OC(＝O)NH(CH2)tNHC(＝O)O-、-NHC(＝O)NH(CH2)tNHC(＝O)NH-、-C(＝O)(CH2)tO-、-C(＝O)(CH2)tC(＝O)O-、-C(＝O)(CH2)tOC(＝O)-、-C(＝O)(CH2)tOC(＝O)O-、-C(＝O)(CH2)tNHC(＝O)O-、-C(＝O)(CH2)tOC(＝O)NH- and-C (=O) (CH2)t NHC (=O) NH-.
1.1.3.B1、B2
In the present invention, B1、B2 is each independently a bond or C1-30 alkylene.
In one embodiment of the invention, B1、B2 is preferably each independently a bond or C1-20 alkylene, more preferably B1、B2 is any of the following:
B1、B2 is independently C1-20 alkylene, specifically B1、B2 is independently any one of methylene, ethylene, propylene, butylene, pentylene, hexylene, heptylene, octylene, nonylene, decylene, undecylene, dodecylene, tridecylene, tetradecylene, pentadecylene, hexadecylene, heptadecylene, octadecylene, nonadecylene and eicosylene, more preferably B1、B2 is independently C5-12 alkylene;
in case (2), B1、B2 is a bond and the other is a C1-20 alkylene group.
1.1.4.R1、R2
In the present invention, R1、R2 are each independentlyC1-30 aliphatic hydrocarbon group or C1-30 aliphatic hydrocarbon derivative residue, and at least one of R1、R2 isWherein t is an integer from 0 to 12, and each Re、Rf is independently any one of C1-C15 alkyl, C2-C15 alkenyl, and C2-C15 alkynyl.
In one embodiment of the present invention, the C1-30 aliphatic hydrocarbon group is preferably a linear alkyl group, a branched alkyl group, a linear alkenyl group, a branched alkenyl group, a linear alkynyl group or a branched alkynyl group, and when the C1-30 aliphatic hydrocarbon group is a branched alkyl group, a branched alkenyl group or a branched alkynyl group, it is represented byThe C1-30 fatty hydrocarbon derivative residue isWherein t is an integer from 0to 12, t1、t2 is each independently an integer from 0to 5, t3、t4 is each independently 0 or 1 and not both 0, wherein Re、Rf is each independently any one of C1-C15 alkyl, C2-C15 alkenyl and C2-C15 alkynyl.
In a specific embodiment of the present invention, preferably the C1-30 aliphatic hydrocarbon group or C1-30 aliphatic hydrocarbon derivative residue is selected from any of the following structures:
in one embodiment of the present invention, theRe、Rf in (a) is independently C1-15 alkyl, selected from any one of methyl, ethyl, propyl, butyl, amyl, hexyl, heptyl, octyl, nonyl and decyl, and a preparation method thereofPreferably selected from any one of the following structures:
1.1.5.R3
in the present invention, R3 is a hydrogen atom 、-Rd、-ORd、-NRdRd、-SRd、-(C＝O)Rd、-(C＝O)ORd、-O(C＝O)Rd、-O(C＝O)ORd orWherein each occurrence of Rd is independently C1-12 alkyl, two Rd groups in NRdRd may be joined to form a ring, G1 is a terminal branching group of k+1 valence, j is 0 or 1, F contains a functional group R01, when j is 0, G1 is absent, when j is 1, G1 gives k F, and k is an integer from 2 to 8.
In one embodiment of the invention, R3 is preferably, independently at each occurrence, a hydrogen atom, Rd、ORd、-(C＝O)Rd-、-(C＝O)ORd、-O(C＝O)Rd、-O(C＝O)ORd andMore preferably contains any one of a hydrogen atom, an alkyl group, an alkoxy group, an alcoholic hydroxyl group, a protected alcoholic hydroxyl group, a thiol hydroxyl group, a protected thiol hydroxyl group, a carboxyl group, a protected carboxyl group, an amino group, a protected amino group, an aldehyde group, a protected aldehyde group, an ester group, a carbonate group, a carbamate group, a succinimidyl group, a maleimide group, a protected maleimide group, a dimethylamino group, an alkenyl group, an alkenoate group, an azido group, an alkynyl group, she Suanji, a rhodamine group and a biotin group, and still more preferably contains H、-(CH2)tOH、-(CH2)tSH、-OCH3、-OCH2CH3、-(CH2)tNH2、-(CH2)tC(＝O)OH、-C(＝O)(CH2)tC(＝O)OH、-C(＝O)CH3、-(CH2)tN3、-C(＝O)CH2CH3、-C(＝O)OCH3、-OC(＝O)OCH3、-C(＝O)OCH2CH3、-OC(＝O)OCH2CH3、-(CH2)tN(CH3)2、-(CH2)tN(CH2CH3)2、-(CH2)tCHO、Any of which, wherein each occurrence of Rd is independently C1-12 alkyl.
1.1.6. Specific general structural formulas are exemplified
In a specific embodiment of the present invention, when X in the general structural formula (1) is N, the structure of the cationic lipid of the present invention preferably satisfies any one of the following structural formulas:
Wherein in formulae (2-39) to (2-48), R1 is, independently at each occurrence, a C1-30 aliphatic hydrocarbon or C1-30 aliphatic hydrocarbon derivative residue, and R2 is, independently at each occurrence, eachThe definitions of s, L3、B1、B2、R3、R1 and R2 are as described in the general formula (1) and are not repeated here.
1.1.7. Specific structural examples
Some embodiments of the invention result in cationic lipids having the structure shown below, including but not limited to any of the following structures:
2. preparation of cationic lipids
In the present invention, any of the foregoing cationic lipids can be prepared by methods including, but not limited to, the following:
2.1. Method 1:
The method comprises the steps of firstly, reacting a small molecule A-1 with a small molecule A-2 to generate a small molecule intermediate A-3 containing a divalent connecting group L1, a reactive group FN at one end and R1 at one end, wherein the small molecule A-1 contains a reactive gene F1, the small molecule A-2 contains a pair of F2 and FN,F2 containing heterofunctional groups as the reactive groups, and the pair of F2 and FN,F2 can react with the F1 to generate a divalent connecting group L1,FN as the reactive group capable of reacting with amino or secondary amino, and the reactive groups are preferably-OMs, -OTs, -CHO, -F, -Cl, -Br;
Carrying out alkylation reaction on a two-molecule small molecule intermediate A-3 and a primary ammonia derivative A-4 containing a nitrogen source end group to obtain cationic lipid A-5, wherein the R3' end contains a reactive group R01 or contains a micro-variation form of R01, and the micro-variation form refers to a group which can be converted into R01 through any chemical process of deprotection, salt complexation and decomplexing, ionization, protonation, deprotonation and change of leaving groups;
When R3 'is equal to R3, the obtained structure A-5' corresponds to the structure shown in the general formula (1);
When R3 'is not equal to R3, carrying out terminal micro-modification on the A-5' to obtain a structure shown in a general formula (1) corresponding to the A-5, wherein the terminal micro-modification is selected from the following chemical reactions of deprotection, salt complexation and decomplexing, ionization, protonation, deprotonation and leaving group change, wherein R1 and R2 are the same, B1 and B2 are the same, and L1 and L2 are the same;
Wherein, the definitions of L1、L2、L3、B1、B2、R3、R1 and R2 are the same as those in the general formula (1), and the description thereof is omitted.
Each of the aforementioned small molecule raw materials A-1, A-2, A-4, etc. may be obtained by purchase or by autonomous synthesis, for example, the small molecule A-1 in example 1.1 isWhich can be achieved byIs obtained by autonomous synthesis of raw materials.
Step one
Step two
2.2. Method 2:
step one, reacting a small molecule B-1 with a small molecule B-2 to generate a small molecule intermediate B-3 containing a divalent connecting group L1, a hydroxyl group at one end and R1 at one end, wherein the small molecule B-1 contains a reactive gene F1, and the small molecule B-2 contains a pair F2 containing an hetero-functional group and a hydroxyl group (OH), and F2 is a reactive group and can react with F1 to generate a divalent connecting group L1;
Oxidizing the hydroxyl of the small molecule intermediate B-3 into an aldehyde group to obtain a small molecule intermediate B-4 containing the aldehyde group, wherein B1' is alkylene which is one methylene less than B1;
Step three, carrying out addition reaction on two molecules of small molecule intermediates B-4 containing aldehyde groups and primary ammonia derivatives B-5 containing nitrogen source end groups to obtain cationic lipid B-6', wherein the R3' end contains a reactive group R01 or contains a micro-variation form of R01, wherein the micro-variation form refers to a group which can be converted into R01 through any chemical process of deprotection, salt complexation and decomplexing, ionization, protonation, deprotonation and change of leaving groups;
when R3 'is equal to R3, the obtained structure B-6' corresponds to the structure shown in the general formula (1);
When R3 'is not equal to R3, carrying out terminal micro-modification on B-6' to obtain a structure shown in a general formula (1) corresponding to B-6, wherein the terminal micro-modification is selected from the following chemical reactions of deprotection, salt complexation and decomplexing, ionization, protonation, deprotonation and leaving group change, R1 and R2 are the same, B1 and B2 are the same, and L1 and L2 are the same;
Wherein, the definitions of L1、L2、L3、B1、B2、R3、R1 and R2 are the same as those in the general formula (1), and the description thereof is omitted.
The small molecule raw materials B-1, B-2, B-5 and the like can be obtained through purchase or autonomous synthesis.
Step one
Step two
Step three
2.3. Method 3:
Step one, reacting a small molecule C-1 with a small molecule C-2 to generate a small molecule intermediate C-3 containing a divalent linking group L1, a reactive group FN at one end and R1 at one end, reacting a small molecule C-1 'with a small molecule C-2' to generate a small molecule intermediate C-3 containing a divalent linking group L2, at one end is a reactive group FNN, a small molecule intermediate C-3' with one end being R2, wherein the small molecule C-1 contains a reactive group F1, the small molecule C-2 contains an hetero functional group which can react with F1 to generate a divalent linking group L1,FN which is a reactive group capable of reacting with an amino group or a secondary amino group, preferably-OMs, -OTs, -CHO, -F, -Cl, Br, a small molecule C-1 'containing a reactive group F3, a small molecule C-2' containing an iso-functional group reactive to F4 and FNN,F4 and capable of reacting with F3 to form a divalent linking group L2;FNN as a reactive group capable of reacting with an amino or secondary amino group, preferably-OMs, -OTs, -CHO, -F, -Cl, -Br, -COOH, -COCl or an activated carboxyl group, said activated carboxyl group being obtained after activation of the carboxyl group with a carboxyl activating agent;
Step two, carrying out alkylation reaction on a molecule of small molecule intermediate C-3 and a primary ammonia derivative C-4 containing a nitrogen source end group to obtain a secondary amine derivative C-5;
Step three, reacting a secondary amine derivative C-5 with a small molecule intermediate C-3' to generate cationic lipid C-6', wherein the R3 ' end contains a reactive group R01 or contains a micro-variation form of R01, and the micro-variation form refers to a group which can be converted into R01 through any chemical process of deprotection, salt complexation and decomplexing, ionization, protonation, deprotonation and change of leaving groups;
when R3 'is equal to R3, the obtained structure C-6' corresponds to the structure shown in the general formula (1);
When R3 'is not equal to R3, carrying out terminal micro-modification on the C-6' to obtain a structure shown in a general formula (1) corresponding to the C-6, wherein the terminal micro-modification is selected from the following chemical reactions of deprotection, salt complexation and decomplexing, ionization, protonation, deprotonation and leaving group change;
Wherein, the definitions of L1、L2、L3、B1、B2、R3、R1 and R2 are the same as those in the general formula (1), and the description thereof is omitted.
The aforementioned small molecule raw materials C-1, C-1', C-2', C-4, etc. can be obtained by purchase or by autonomous synthesis, for example, the small molecule C-1 in example 6, S6-1 isCan be obtained by purchase or by autonomous synthesis.
Step one
Step two
Step three
R1 in the reaction raw material R1-F1 in the aforementioned preparation method can be etherified aliphatic hydrocarbon derivative residueWherein each occurrence of t is independently an integer from 0 to 12, and Re、Rf is independently any one of C1-C15 alkyl, C2-C15 alkenyl, and C2-C15 alkynyl. More specifically, R1-F1 may beCommercially available, or may be synthesized autonomously by aldol addition, e.g. one moleculeIs added with two molecules Re -OH to obtainRe and Rf are the same at this time, R1-F1 may also beCan be obtained by purchase or synthesized autonomouslyIs obtained by reaction with the relevant alkylating agent, the alkylating agent is preferably a halide, e.gCan be obtained by deprotection after the reaction of glycerol with one molecule of TBS for protecting hydroxyl and two molecules of bromohexane.
2.3. Method 4:
Reacting a trifunctional small molecule D-1 containing two identical reactive groups F5 and R3 ' with two molecules of D-2 to generate cationic lipid D-3', wherein the small molecule D-2 contains a reactive group F6 and can react with F5 to generate a divalent linking group L1 or a micro-variation form containing a reactive group R01 or containing R01 at the end of L2,R3 ', wherein the micro-variation form refers to a group which can be converted into R01 through any one of the chemical processes of deprotection, salt complexation and decomplexing, ionization, protonation, deprotonation and change of leaving groups;
When R3 'is equal to R3, the obtained structure D-3' corresponds to the structure shown in the general formula (1);
When R3 'is not equal to R3, carrying out terminal micro-modification on D-3' to obtain a structure shown in a general formula (1) corresponding to D-3, wherein the terminal micro-modification is selected from the following chemical reactions of deprotection, salt complexation and decomplexing, ionization, protonation, deprotonation and leaving group change, R1 and R2 are the same, and L1 and L2 are the same;
Wherein X, L1、L2、、L3、B1、B2、R3、R1 and R2 are as defined in formula (1), and are not described in detail herein.
The small molecule raw materials D-1 and D-2 can be obtained through purchase or autonomous synthesis.
2.5. Description of related raw materials and/or procedures in the preparation Process
2.5.1. "Protection" and "deprotection" of related groups during reaction "
In the present invention, the "protection" and "deprotection" processes of the relevant groups are also involved in the reaction process. To prevent the functional group from affecting the reaction, the functional group is usually protected. When the number of functional groups is 2 or more, only the target functional group is selectively reacted, and thus other functional groups are protected. The protecting group not only stably protects the functional group to be treated, but also needs to be easily removed as needed. It is therefore important in organic synthesis to deprotect under appropriate conditions only the protecting group bonded to the specified functional group.
In the present invention, the "carboxyl protecting group" means a protecting group which can be converted into a carboxyl group by a deprotection reaction of the carboxyl protecting group by hydrolysis. The carboxyl protecting group is preferably an alkyl group (e.g., methyl, ethyl, t-butyl) or an aralkyl group (e.g., benzyl), more preferably t-butyl (tBu), methyl (Me) or ethyl (Et). In the present invention, the "protected carboxyl group" means a group formed by protecting a carboxyl group with a suitable carboxyl protecting group, and preferably methoxycarbonyl, ethoxycarbonyl, t-butoxycarbonyl, and benzyloxycarbonyl. The carboxyl protecting groups can be removed hydrolytically with acid or base catalysis, occasionally with elimination by pyrolysis, for example tert-butyl can be removed under mildly acidic conditions and benzyl can be removed by hydrogenolysis. The reagent for removing the carboxyl protecting group is selected from TFA, H2 O, liOH, naOH, KOH, meOH, etOH and combinations thereof, preferably a combination of TFA and H2 O, a combination of LiOH and MeOH, or a combination of LiOH and EtOH. The protected carboxyl group is deprotected to yield the corresponding free acid, said deprotection being carried out in the presence of a base, said base and said free acid formed by said deprotection forming a pharmaceutically acceptable salt.
In the present invention, the "amino protecting group" includes all groups which can be used as protecting groups for general amino groups, for example, arylC1-6 alkyl, C1-6 alkoxyC1-6 alkyl, C1-6 alkoxycarbonyl, aryloxycarbonyl, C1-6 alkylsulfonyl, arylsulfonyl, silyl and the like. The amino protecting group is preferably Boc t-butoxycarbonyl, moz p-methoxybenzyloxycarbonyl or Fmoc 9-fluorenylmethoxycarbonyl. The reagent for removing the amino protecting group is selected from TFA, H2 O, liOH, meOH, etOH and combinations thereof, preferably a combination of TFA and H2 O, a combination of LiOH and MeOH, or a combination of LiOH and EtOH. The reagent for removing Boc protecting group is TFA or HCl/EA, preferably TFA. The deprotection agent used for Fmoc protecting group removal was a 20% piperidine in N, N-Dimethylformamide (DMF).
In the present invention, the hydroxyl group protected by the hydroxyl protecting group is not particularly limited, and may be, for example, a hydroxyl group such as an alcoholic hydroxyl group or a phenolic hydroxyl group. The amino group of the amino protecting group is not particularly limited, and may be derived from, for example, a primary amine, a secondary amine, a diamine, an amide, or the like. Amino groups in the present invention are not particularly limited and include, but are not limited to, primary amino groups, secondary amino groups, tertiary amino groups, quaternary ammonium ions.
In the present invention, deprotection of the protected hydroxyl group is related to the type of hydroxyl protecting group. The type of the hydroxyl protecting group is not particularly limited, and examples of the protecting of the terminal hydroxyl group by benzyl, silyl ether, acetal and tert-butyl are as follows:
deprotection of benzyl groups
Benzyl deprotection can be achieved by hydrogenation of the hydrogenation reducing agent and the hydrogen donor, and the water content in the reaction system should be less than 1%, so that the reaction can be smoothly carried out.
The hydrogenation reduction catalyst is not limited, and palladium and nickel are preferable, but a carrier is not limited, but alumina or carbon is preferable, and carbon is more preferable. The palladium is used in an amount of 1 to 100% by weight of the protected hydroxyl compound, preferably 1 to 20% by weight of the protected hydroxyl compound.
The reaction solvent is not particularly limited as long as both the raw material and the product can be a solvent, but methanol, ethanol, ethyl acetate, tetrahydrofuran, acetic acid are preferable, and methanol is more preferable. The hydrogen donor is not particularly limited, but hydrogen gas, cyclohexene, 2-propanol, ammonium formate and the like are preferable. The reaction temperature is preferably 25 to 40 ℃. The reaction time is not particularly limited, and the reaction time is inversely related to the amount of the catalyst, preferably 1 to 5 hours.
Deprotection of acetals and ketals
The acetal or ketal compounds for such hydroxyl protection are preferably ethyl vinyl ether, tetrahydropyran, acetone, 2-dimethoxypropane, benzaldehyde, etc. Whereas deprotection of such acetals and ketals is achieved under acidic conditions, the solution pH is preferably from 0 to 4. The acid is not particularly limited, but acetic acid, phosphoric acid, sulfuric acid, hydrochloric acid, nitric acid are preferable, and hydrochloric acid is more preferable. The reaction solvent is not particularly limited as long as it can dissolve the reactants and products, and water is preferable. The reaction temperature is preferably from 0 to 30 ℃.
Deprotection of silyl ethers
Compounds useful for such hydroxyl protection include trimethylsilyl ether, triethylsilyl ether, dimethyl t-butylsilyl ether, t-butyldiphenylsilyl ether, and the like. The deprotection of such silyl ethers is carried out by a fluoride ion-containing compound, preferably tetrabutylammonium fluoride, tetraethylammonium fluoride, hydrofluoric acid, potassium fluoride, more preferably tetrabutylammonium fluoride, potassium fluoride. The amount of the fluorine-containing agent is 5 to 20 times, preferably 8 to 15 times, the molar equivalent of the protected hydroxyl group, and if the amount of the fluorine-containing agent is less than 5 times the molar equivalent of the protected hydroxyl group, the deprotection becomes incomplete, and when the amount of the deprotecting agent is more than 20 times the molar equivalent of the protected hydroxyl group, an excessive amount of the agent or compound causes trouble in purification and may be mixed in the subsequent step, thereby causing side reactions. The reaction solvent is not particularly limited as long as it can dissolve the reactants and products, and aprotic solvents are preferable, and tetrahydrofuran and methylene chloride are more preferable. The reaction temperature is preferably 0 to 30 ℃, and when the temperature is lower than 0 ℃, the reaction speed is low, and the protecting group cannot be completely removed.
Deprotection of tert-butyl group
The deprotection of the tert-butyl group is carried out under acidic conditions, the solution pH being preferably from 0 to 4. The acid is not particularly limited, but acetic acid, phosphoric acid, sulfuric acid, hydrochloric acid, nitric acid are preferable, and hydrochloric acid is more preferable. The reaction solvent is not particularly limited as long as it can dissolve the reactants and products, and water is preferable. The reaction temperature is preferably from 0 to 30 ℃.
In the terminal functionalization method, q=0 and q1＝1,Z1 is preferably 1, 2-methylene. When q is other than 0, a and R01 have a linker such as an amino acid, succinyl, etc., the techniques known in the art to produce Z2 or Z1 (including but not limited to alkylation, condensation, click reactions, etc.) can be used and prepared with reference to the linear functionalization schemes described below.
2.5.2. Alkylation reaction
The alkylation reaction according to the invention is preferably a reaction based on the alkylation of hydroxyl, mercapto or amino groups, which in turn corresponds to the formation of ether linkages, thioether linkages, secondary or tertiary amino groups. Examples are as follows:
2.5.2.1. alkylation of substrate alcohol with sulfonate and halogenide
Nucleophilic substitution of a substrate alcohol with a sulfonate derivative, a halide, in the presence of a base, yields an amine intermediate. Wherein the molar equivalent of sulfonate, halide is 1 to 50 times, preferably 1 to 5 times that of the substrate alcohol. When the molar equivalent of the sulfonate or the halogenide is less than 1 time the molar equivalent of the substrate alcohol, the substitution by the reaction is incomplete, and purification is difficult. And when the molar equivalent of sulfonate and halogenide is more than 50 times of that of substrate alcohol, the excessive reagent brings trouble to purification and may be mixed into the subsequent step, thereby causing the increase of the side reaction of the next step and increasing the difficulty of purification.
The resulting product is a mixture of the ether intermediate and excess sulfonate, halide, which may be purified by means of anion exchange resins, osmosis, ultrafiltration, etc. Among them, the anion exchange resin is not particularly limited as long as the target product can be ion-exchanged and adsorbed on the resin, and an ion exchange resin of tertiary amine or quaternary ammonium salt having dextran, agarose, polyacrylate, polystyrene, polydistyrene or the like as a skeleton is preferable. The solvent for permeation and ultrafiltration is not limited, and water or an organic solvent is generally used, and the organic solvent is not particularly limited as long as the product can be dissolved therein, and methylene chloride, chloroform and the like are preferable.
The reaction solvent is not limited, and aprotic solvents such as toluene, benzene, xylene, acetonitrile, ethyl acetate, tetrahydrofuran, chloroform, methylene chloride, dimethyl sulfoxide, dimethylformamide or dimethylacetamide are preferable, and dimethylformamide, methylene chloride, dimethyl sulfoxide or tetrahydrofuran are more preferable.
The base includes an organic base (such as triethylamine, pyridine, 4-dimethylaminopyridine, imidazole or diisopropylethylamine) or an inorganic base (such as sodium carbonate, sodium hydroxide, sodium bicarbonate, sodium acetate, potassium carbonate or potassium hydroxide), preferably an organic base, more preferably triethylamine, pyridine. The molar amount of base is 1 to 50 times, preferably 1 to 10 times, more preferably 3 to 5 times the molar equivalent of sulfonate or halide.
2.5.2.2. Alkylation of substrate amine with sulfonate and halogenide
A. alkylation of substrate amine with sulfonate and halogenide
Nucleophilic substitution of the substrate amine with sulfonate derivatives and halides in the presence of a base yields an amine intermediate. Wherein the molar equivalent of sulfonate and halide is 1 to 50 times, preferably 1 to 5 times that of substrate amine. When the molar equivalent of the sulfonate or the halogenide is less than 1 time the molar equivalent of the substrate amine, the substitution is incomplete, and purification is difficult. And when the molar equivalent of sulfonate and halogenide is more than 50 times of that of substrate amine, the excessive reagent brings trouble to purification and may be mixed into the subsequent step, thereby causing the increase of the side reaction of the next step and increasing the purification difficulty.
The resulting product is a mixture of amine intermediate and excess sulfonate, halide, which may be purified by means of anion exchange resins, osmosis, ultrafiltration, etc. Among them, the anion exchange resin is not particularly limited as long as the target product can be ion-exchanged and adsorbed on the resin, and an ion exchange resin of tertiary amine or quaternary ammonium salt having dextran, agarose, polyacrylate, polystyrene, polydistyrene or the like as a skeleton is preferable. The solvent for permeation and ultrafiltration is not limited, and water or an organic solvent is generally used, and the organic solvent is not particularly limited as long as the product can be dissolved therein, and methylene chloride, chloroform and the like are preferable.
The reaction solvent is not limited, and aprotic solvents such as toluene, benzene, xylene, acetonitrile, ethyl acetate, tetrahydrofuran, chloroform, methylene chloride, dimethyl sulfoxide, dimethylformamide or dimethylacetamide are preferable, and dimethylformamide, methylene chloride, dimethyl sulfoxide or tetrahydrofuran are more preferable.
The base includes an organic base (such as triethylamine, pyridine, 4-dimethylaminopyridine, imidazole or diisopropylethylamine) or an inorganic base (such as sodium carbonate, sodium hydroxide, sodium bicarbonate, sodium acetate, potassium carbonate or potassium hydroxide), preferably an organic base, more preferably triethylamine, pyridine. The molar amount of base is 1 to 50 times, preferably 1 to 10 times, more preferably 3 to 5 times the molar equivalent of sulfonate or halide.
2.5.2.3. Alkylation reaction of substrate amine and aldehyde derivative
The imine intermediate is obtained by reacting substrate amine with aldehyde derivative, and then the intermediate is obtained under the action of reducing agent. Wherein the molar equivalent of the aldehyde derivative is 1 to 20 times, preferably 1 to 2 times, more preferably 1 to 1.5 times that of the substrate amine. When the molar equivalent of the aldehyde derivative is more than 20 times that of the substrate amine, an excessive amount of the reagent brings trouble to purification, and may be mixed in the subsequent step, increasing the difficulty of purification. When the molar equivalent of the aldehyde derivative is less than 1 time of the substrate amine, the reaction is incomplete, and the purification difficulty is increased. Wherein, the reaction product can be purified by means of cation exchange resin, permeation, ultrafiltration and the like to obtain an intermediate. The cation exchange resin is not particularly limited as long as it can exchange with a quaternary ammonium cation to achieve a separation effect. The solvent for permeation and ultrafiltration is not limited, and water or an organic solvent is generally used, and the organic solvent is not particularly limited as long as the product can be dissolved therein, and methylene chloride, chloroform and the like are preferable.
The reaction solvent is not limited, and organic solvents such as methanol, ethanol, water, toluene, benzene, xylene, acetonitrile, ethyl acetate, tetrahydrofuran, chloroform, methylene chloride, dimethyl sulfoxide, dimethylformamide, dimethylacetamide, and the like are preferable, and water and methanol are more preferable.
The reducing agent is not particularly limited as long as it can reduce the imine to an amine, and sodium borohydride, lithium aluminum hydride, sodium cyanoborohydride, zn/AcOH and the like are preferable, and sodium cyanoborohydride is more preferable. The amount of the reducing agent to be used is generally 0.5 to 50 times, more preferably 1 to 10 times, the amount of the aldehyde derivative substance.
2.5.3. Trifunctional small molecule D-1
The trifunctional small molecule D-1 contains two identical reactive groups F5 and R3 ', wherein F5 is a reactive group, and the R3' end contains a reactive group R01 or contains a micro-variation of R01, wherein the micro-variation refers to a group which can be converted into R01 through any chemical process of deprotection, salt complexation and decomplexing, ionization, protonation, deprotonation and change of leaving groups.
In particular, the trifunctional small molecule D-1 includes, but is not limited to, any of the following structures:
Etc. also include the cases where the relevant groups in the foregoing trifunctional small molecules are protected, e.gIt can also be the case in which the amino group is protected, i.e
2.5.4. Linear functionalization of terminal ends
The method of terminal linear functionalization is not particularly limited, and is related to the type of final functional group or protected form thereof.
Linear functionalization of the terminal hydroxyl group, i.e. starting from the terminal hydroxyl group of the compound A-5', by functionalization to give other functional groups or protected forms thereof-L3-R3, is carried out by the preparation process described in paragraphs [0960] to [1205] of the document CN 104530417A.
In the invention, raw materials used in each preparation method can be obtained by purchase or self-synthesis.
The intermediates and end products prepared in the present invention may be purified by purification methods including, but not limited to, extraction, recrystallization, adsorption treatment, precipitation, reverse precipitation, thin film dialysis, or supercritical extraction. Characterization of the structure, molecular weight, etc. of the final product can be confirmed by characterization methods including, but not limited to, nuclear magnetism, electrophoresis, ultraviolet-visible spectrophotometry, FTIR, AFM, GPC, HPLC, MALDI-TOF, circular dichroism, etc.
3.1. Cationic liposome
In the invention, a cationic liposome contains any cationic lipid with a structure shown as a general formula (1).
In one embodiment of the present invention, it is preferable that the cationic liposome contains one or more of neutral lipid, steroid lipid and pegylated lipid in addition to the cationic lipid having the structure represented by the general formula (1), and more preferably contains three lipids of neutral lipid, steroid lipid and pegylated lipid at the same time. The neutral lipid is preferably a phospholipid.
In one embodiment of the present invention, the neutral lipids in the cationic liposome preferably include, but are not limited to, 1, 2-dioleoyl-sn-glycero-3-phosphorylcholine (DLPC), 1, 2-dimyristoyl-sn-glycero-phosphorylcholine (DMPC), 1, 2-dioleoyl-sn-glycero-3-phosphorylcholine (DOPC), 1, 2-dipalmitoyl-sn-glycero-3-phosphorylcholine (DPPC), 1, 2-distearoyl-sn-glycero-3-phosphorylcholine (DSPC), 1, 2-dioleoyl-sn-glycero-phosphorylcholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphorylcholine (POPC), 1, 2-dioleoyl-sn-glycero-3-phosphorylcholine (18:0 Dietherepc), 1-oleoyl-2-sterolylhemisuccinyl-sn-glycero-3-phosphorylcholine (DPPC), 1, 2-distearoyl-sn-glycero-3-phosphorylcholine (DSPC), 1, 2-dioleoyl-sn-glycero-3-phosphorylcholine (864), 1, 2-dioleoyl-sn-glycero-3-phosphorylcholine (POPC), 1, 2-dioleoyl-glycero-3-phosphorylcholine (SDP) and hexa-glycero-3-phosphorylcholine (SDP) are included 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine, 1, 2-didodecyloyl-sn-glycero-3-phosphoethanolamine, 1, 2-dioleoyl-sn-glycero-3-phospho-rac- (1-glycero-sodium salt (DOPG), dioleoyl phosphatidylserine (DOPS), dipalmitoyl phosphatidylglycerol (DPPG), palmitoyl Phosphatidylethanolamine (PE), distearoyl-phosphatidylethanolamine (DSPE), palmitoyl phosphatidylethanolamine (DSPE), dioleoyl-phosphatidylethanolamine (DPPE), stearoyl-phosphatidylethanolamine (DPPC), stearoyl-phosphatidylethanolamine (SOtidyl-phosphatidylethanolamine, stearoyl-2-phosphatidylethanolamine (PSOtidyl-PE), stearoyl-phosphatidylethanolamine (SOP), stearoyl-Phosphatidylethanolamine (PSE), any one of palmitoyl phosphatidylcholine, lysophosphatidylcholine, and Lysophosphatidylethanolamine (LPE) and a composition thereof.
In a specific embodiment of the present invention, the steroid lipid in the cationic liposome is preferably any one of cholesterol, fecal sterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, lycorine, ursolic acid, alpha-tocopherol, and mixtures thereof.
In a specific embodiment of the present invention, the pegylated lipid in the cationic liposome is preferably any one of polyethylene glycol-1, 2-dimyristate glyceride (PEG-DMG), polyethylene glycol-distearoyl phosphatidylethanolamine (PEG-DSPE), PEG-cholesterol, polyethylene glycol-diacylglycerol (PEG-DAG), polyethylene glycol-dialkoxypropyl (PEG-DAA), specifically polyethylene glycol 500-dipalmitoyl phosphatidylcholine, polyethylene glycol 2000-dipalmitoyl phosphatidylcholine, polyethylene glycol 500-stearoyl phosphatidylethanolamine, polyethylene glycol 2000-distearoyl phosphatidylethanolamine, polyethylene glycol 500-1, 2-oleoyl phosphatidylethanolamine, polyethylene glycol 2000-1, 2-oleoyl phosphatidylethanolamine and polyethylene glycol 2000-2, 3-dimyristoyl phosphatidylglycerol (PEG-DMG).
In one embodiment of the present invention, the structure of the pegylated lipid in the cationic liposome is preferably as shown in the general formula (2):
or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof,
Wherein each L7、L8 is independently a bond or a divalent linker selected from any one of -O(C＝O)-、-(C＝O)O-、-O(C＝O)O-、-C(＝O)-、-O-、-S-、-C(＝O)S-、-SC(＝O)-、-NRcC(＝O)-、-C(＝O)NRc-、-NRcC(＝O)NRc-、-OC(＝O)NRc-、-NRcC(＝O)O-、-SC(＝O)NRc- and-NRc C (=o) S-, wherein each occurrence of Rc is independently a hydrogen atom or a C1-12 alkyl group;
L3 is a bond or a divalent linking group, and when a divalent linking group is selected from any one, any two or a combination of any two or more of L4、L5 and Z divalent linking groups, more preferably any one of-L4-、-Z-L4-Z-、-L4-Z-L5-、-Z-L4-Z-L5 -and-L4-Z-L5 -Z-, wherein L4、L5 is a carbon chain linking group, each independently -(CRaRb)t-(CRaRb)o-(CRaRb)p-,t、o、p is an integer of 0 to 12, and t, O, and p are not simultaneously 0, each independently Ra and Rb is a hydrogen atom or a C1-12 alkyl group, each independently Z is any one of -(C＝O)-、-O(C＝O)-、-(C＝O)O-、-O(C＝O)O-、-O-、-S-、-C(＝O)S-、-SC(＝O)-、-NRcC(＝O)-、-C(＝O)NRc-、-NRcC(＝O)NRc-、-OC(＝O)NRc-、-NRcC(＝O)O-、-SC(＝O)NRc- and-NRc C (=O) S-, wherein each independently Rc is H or a C1-12 alkyl group;
each B3、B4 is independently a bond or C1-12 alkylene;
each R1、R2 is independently a C1-30 aliphatic hydrocarbon group;
R is a hydrogen atom, an alkyl group, an alkoxy group, - (C=O) Rd、-(C＝O)ORd、-O(C＝O)Rd、-O(C＝O)ORd orWherein Rd is C1-12 alkyl, G1 is a k+1-valent terminal branching group, j is 0 or 1, F contains functional groups, G1 is absent when j is 0, G1 draws k F when j is 1, and k is an integer of 2-8;
A is- (CRaRb)s O-or-O (CRaRb)s -, wherein s is 2,3 or 4, Ra and Rb are each independently a hydrogen atom or a C1-12 alkyl group);
n1 is an integer from 20 to 250;
the alkyl, alkylene, alkoxy, aliphatic hydrocarbon groups are each independently substituted or unsubstituted.
In a specific embodiment of the present invention, the structure of the pegylated lipid in the cationic liposome is represented by general formula (2) and is selected from any one of the following structural formulas:
in a specific embodiment of the present invention, it is preferred that any of the aforementioned cationic liposomes comprises 20 to 80% of the cationic lipid represented by formula (1), 5 to 15% of the neutral lipid, 25 to 55% of the steroid lipid and 0.5 to 10% of the pegylated lipid, said percentages being the mole percentage of each lipid based on the total lipid in the solution comprising the solvent.
In a specific embodiment of the present invention, it is preferred that the cationic lipid comprises 30-65% by mole of the total lipid in the solvent-containing solution in any of the foregoing cationic liposomes, more preferably about any of 35%, 40%, 45%, 46%, 47%, 48%, 49%, 50%, 55%.
In a specific embodiment of the present invention, preferably, the neutral lipid comprises 7.5-13% by mole of the total lipid in the solvent-containing solution in any of the foregoing cationic liposomes, more preferably about any one of 8%, 9%, 10%, 11%, 12%.
In a specific embodiment of the present invention, it is preferred that the mole percentage of steroid lipids in the solution comprising solvent in any of the foregoing cationic liposomes is from 35 to 50%, more preferably about any of 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%.
In a specific embodiment of the present invention, it is preferred that the pegylated lipid comprises 0.5-5%, preferably 1-3%, more preferably about any of 1.5%, 1.6%, 1.7%, 1.8%, 1.9% by mole of the total lipid in the solvent-containing solution in any of the foregoing cationic liposomes.
3.2. Preparation of cationic liposomes
In the present invention, the cationic liposome may be prepared by methods including, but not limited to, a thin film dispersion method, an ultrasonic dispersion method, a reverse phase evaporation method, a freeze-drying method, a freeze-thawing method, a multiple emulsion method and/or an injection method, a microfluidic method, and preferably a thin film dispersion method or an injection method.
4.1. Cationic liposome pharmaceutical compositions
In one embodiment of the invention, a cationic liposome pharmaceutical composition comprises any one of the cationic liposome and a drug, wherein the cationic liposome comprises any one of the cationic liposome with a structure shown as a general formula (1), and the drug comprises, but is not limited to, a nucleic acid drug, a genetic vaccine, an anti-tumor drug, a small molecule drug, a polypeptide drug, a protein drug and the like.
In a specific embodiment of the invention, the cationic liposome pharmaceutical composition is prepared by a simple mixing method or a microfluidic method, specifically, cationic lipid, neutral lipid, steroid lipid and PEGylated lipid are dissolved in an organic phase according to a certain mole percentage to obtain an organic phase solution, a drug (therapeutic agent or preventive agent) is added into an aqueous phase according to a certain N/P ratio to obtain an aqueous phase solution, the organic phase solution and the aqueous phase solution are mixed (microfluidic mixing or simple mixing) according to a proper volume ratio, and the cationic liposome pharmaceutical composition is obtained by post-treatment and purification.
In a specific embodiment of the present invention, the preferred drug in the cationic liposome pharmaceutical composition is a nucleic acid drug selected from any one of RNA, DNA, antisense nucleic acid, plasmid, mRNA (messenger RNA), interfering nucleic acid, aptamer, miRNA inhibitor (antagomir), microrna (miRNA), ribozyme and small interfering RNA (siRNA), preferably any one of RNA, miRNA and siRNA.
In one embodiment of the present invention, the cationic liposome pharmaceutical composition is preferably used as a drug, including, but not limited to, anti-tumor agents, antiviral agents, antifungal agents, and vaccines.
In a specific embodiment of the invention, the drug in the cationic liposome pharmaceutical composition is a nucleic acid drug, and the N/P ratio of the cationic lipid to the nucleic acid is (0.5-20): 1, more preferably (1-10): 1, still more preferably 2:1, 4:1, 6:1 or 10:1.
In a specific embodiment of the invention, the water phase for dissolving the nucleic acid medicine is preferably deionized water, ultrapure water, phosphate buffer or physiological saline, more preferably phosphate buffer or citrate buffer, most preferably citrate buffer, and the cationic liposome is preferably (0.05-20) g/100 mL, more preferably (0.1-10) g/100 mL, most preferably (0.2-5) g/100 mL.
5.1A cationic Liposome pharmaceutical composition formulation
In the present invention, a cationic liposome pharmaceutical composition preparation contains any one of the cationic liposome pharmaceutical compositions described above and a pharmaceutically acceptable diluent or excipient, wherein the diluent or excipient is preferably any one of deionized water, ultrapure water, phosphate buffer and physiological saline, more preferably phosphate buffer or physiological saline, and most preferably physiological saline.
The preparation of cationic lipids, cationic liposomes, cationic liposome nucleic acid pharmaceutical compositions, and the testing of the bioactivity of cationic liposome nucleic acid pharmaceutical compositions are further described below in connection with some specific examples. The present invention will be described in further detail with reference to specific examples, which are not intended to limit the scope of the invention. In examples where cationic lipids are prepared, the final product is characterized by nuclear magnetism, or molecular weight is confirmed by MALDI-TOF.
Example 1:
EXAMPLE 1.1 cationic lipid (E1-1)
Corresponding to the general formula (1), R1、R2 in E1-1 areB1、B2 is hexylene, L1、L2 is ester (-C (=O) O-), X is N, L3 is butylene, R3 is hydroxy, and the total molecular weight is about 824Da.
The preparation process is as follows:
Step a to the compound N-hexyloctylamine (S1-1, 5.33g,25.0 mmol) was added 100mL of anhydrous dichloromethane and dissolved by stirring at room temperature. Potassium carbonate (K2CO3, 5.95g,50.0 mmol), 3-methanesulfonyloxy propionic acid (S1-2, 0.84g,5.0 mmol) and tetra-n-butylammonium bromide (0.19 g,0.6 mmol) were added in this order, and the reaction was stirred at room temperature for 72 hours. After the reaction, 50mL of water was added, stirred and mixed, the pH was adjusted to 5-7, extracted twice with dichloromethane (50 mL x 2), the organic phases were combined, backwashed once with saturated aqueous sodium chloride (50 mL), the organic phases were retained, dried over anhydrous sodium sulfate, filtered, and the filtrate concentrated to give crude compound S1-3. Purification by column chromatography, concentration and pumping-out of an oil pump gave 3- (N-hexyloctylamino) propionic acid (S1-3, 2.20 g).
Step b to a round bottom flask containing S1-3 (2.00 g,7.0 mmol), 6-bromo-n-hexanol (S1-4, 1.51g,8.4 mmol) and 4- (dimethylamino) pyridine (DMAP, 0.21g,1.8 mmol) in dichloromethane (50 mL) was added dicyclohexylcarbodiimide (DCC, 3.17g,15.4 mmol) under an argon atmosphere and reacted at room temperature for 16h. After the completion of the reaction, the precipitate was removed by filtration, and the filtrate was concentrated, and the obtained residue was purified by silica gel column chromatography to obtain brominated ester S1-5 (2.55 g).
Step c-under nitrogen, the compound 4-amino-1-butanol (S1-6, 0.18g,2.0 mmol) was dissolved in acetonitrile (50 mL) and S1-5 (2.24 g,5.0 mmol) and N, N-diisopropylethylamine (DIPEA, 0.36g,4.0 mmol) were added sequentially with slow stirring and reacted at room temperature for about 20h. After the completion of the reaction, the reaction mixture was concentrated and dissolved in methylene chloride, and then extracted with 0.6M hydrochloric acid/10% sodium chloride solution and saturated sodium bicarbonate solution in this order, and the organic phases were combined, dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by column chromatography to give cationic lipid E1-1 (1.32 g). The main data of nuclear magnetic hydrogen spectrum of E1-1 is shown below ：1H NMR(400MHz,CDCl3)δ:4.06(t,4H),3.64-3.61(m,2H),3.24(t,4H),3.03(t,4H),3.02-2.81(m,14H),1.80-1.21(m,60H),0.87(t,12H). through MALDI-TOF test, and the molecular weight of E1-1 is 823.76Da.
EXAMPLE 1.2 cationic lipid (E1-2)
Corresponding to the general formula (1), R1、R2 in E1-2 areB1、B2 is hexylene, L1、L2 is ester (-C (=O) O-), X is N, L3 is ethylene, R3 is hydroxy, and the total molecular weight is about 796Da.
The preparation process is as follows:
The compound 2-amino-1-ethanol (S1-7, 0.12g,2.0 mmol) was dissolved in acetonitrile (50 mL) under nitrogen, S1-5 (2.24 g,5.0 mmol) and DIPEA (0.36 g,4.0 mmol) were added sequentially with slow stirring and reacted at room temperature for about 20h with stirring. After the completion of the reaction, the reaction mixture was concentrated and dissolved in methylene chloride, and then extracted with 0.6M hydrochloric acid/10% sodium chloride solution and saturated sodium bicarbonate solution in this order, and the organic phases were combined, dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by column chromatography to give cationic lipid E1-2 (1.27 g). The main data of nuclear magnetic hydrogen spectrum of E1-2 is shown below ：1H NMR(400MHz,CDCl3)δ:4.04(t,4H),3.86-3.78(m,2H),3.23(t,4H),3.03(t,4H),3.02-2.81(m,14H),1.81-1.22(m,56H),0.87(t,12H). through MALDI-TOF test, and the molecular weight of E1-2 is 795.75Da.
EXAMPLE 1.3 cationic lipid (E1-3)
Corresponding to the general formula (1), R1、R2 in E1-3 areB1、B2 is butylene, L1、L2 is ester (-C (=O) O-) and X is N, L3 is butylene, R3 is hydroxy, and the total molecular weight is about 768Da.
The preparation process is as follows:
Step a to a round bottom flask containing S1-3 (2.85 g,10.0 mmol), 4-bromo n-butanol (S1-8, 1.82g,12.0 mmol) and DMAP (0.31 g,2.5 mmol) in dichloromethane (150 mL) was added DCC (4.53 g,22.0 mmol) under argon atmosphere and reacted at room temperature for 16h. After the completion of the reaction, the precipitate was removed by filtration, and the filtrate was concentrated, and the obtained residue was purified by silica gel column chromatography to obtain brominated ester S1-9 (3.59 g).
Step b Compounds S1-6 (0.18 g,2.0 mmol) were dissolved in acetonitrile (50 mL) under nitrogen and S1-9 (2.10 g,5.0 mmol) and DIPEA (0.36 g,4.0 mmol) were added sequentially with slow stirring and reacted at room temperature for about 20h. After the completion of the reaction, the reaction mixture was concentrated and dissolved in methylene chloride, and then extracted with 0.6M hydrochloric acid/10% sodium chloride solution and saturated sodium bicarbonate solution in this order, and the organic phases were combined, dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by column chromatography to give cationic lipid E1-3 (1.23 g). The main data of nuclear magnetic hydrogen spectrum of E1-3 is shown below ：1H NMR(400MHz,CDCl3)δ:4.07(t,4H),3.64-3.61(m,2H),3.24(t,4H),3.03(t,4H),3.02-2.81(m,14H),1.81-1.22(m,52H),0.87(t,12H). through MALDI-TOF test, and the molecular weight of E1-3 is 767.70Da.
EXAMPLE 2 cationic lipid (E2-1)
Corresponding to the general formula (1), R1、R2 in E2-1 areB1、B2 is hexylene, L1、L2 is ester (-C (=O) O-), X is N, L3 is butylene, R3 is hydroxy, and the total molecular weight is about 768Da.
The preparation process is as follows:
Step a Compound S1-1 (2.57 g,12.0 mmol) was dissolved in dichloromethane (50 mL), then 6-bromohexyl-N-succinimidyl carbonate (S2-1, 3.22g,10.0 mmol) and triethylamine (TEA, 1.10mL,15.0 mmol) were added sequentially and the reaction was stirred at room temperature overnight. After the reaction is finished, the reaction solution is concentrated to obtain a crude product. Purification by column chromatography, concentration and pumping-out with an oil pump gave bromoesterified compound S2-2 (3.31 g).
Step b Compound S1-6 (0.18 g,2.0 mmol) was dissolved in acetonitrile (50 mL) under nitrogen, S2-2 (2.10 g,5.0 mmol) and DIPEA (0.36 g,4.0 mmol) were added sequentially with slow stirring and reacted at room temperature for about 20h. After the completion of the reaction, the reaction mixture was concentrated and dissolved in methylene chloride, and extracted with 0.6M hydrochloric acid/10% sodium chloride solution and saturated sodium bicarbonate solution in this order, and the organic phases were combined, dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by column chromatography to give cationic lipid E2-1 (1.25 g). The main data of nuclear magnetic hydrogen spectrum of E2-1 is shown below ：1H NMR(400MHz,CDCl3)δ:4.05(t,4H),3.65-3.61(m,2H),3.22-3.06(m,8H),2.91-2.63(m,6H),1.81-1.22(m,60H),0.88(t,12H). through MALDI-TOF test, and the molecular weight of E2-1 is 767.73Da.
EXAMPLE 3 cationic lipid (E3-1)
Corresponding to the general formula (1), R1、R2 in E3-1 areB1、B2 is hexylene, L1、L2 is ester (-C (=O) O-), X is N, L3 is butylene, R3 is hydroxy, and the total molecular weight is about 824Da.
The preparation process is as follows:
Step a Compound S3-1 (2.89 g,12.0 mmol) was dissolved in dichloromethane (50 mL) and then S2-1 (3.22 g,10.0 mmol) and TEA (1.10 mL,15.0 mmol) were added sequentially and the reaction stirred at room temperature overnight. After the reaction is finished, the reaction solution is concentrated to obtain a crude product. Purification by column chromatography, concentration and oil pump drainage gave the compound bromoesterified S3-2 (3.49 g).
Step b Compound S1-6 (0.18 g,2.0 mmol) was dissolved in acetonitrile (50 mL) under nitrogen, S3-2 (2.24 g,5.0 mmol) and DIPEA (0.36 g,4.0 mmol) were added sequentially with slow stirring and reacted at room temperature for about 20h. After the completion of the reaction, the reaction mixture was concentrated and dissolved in methylene chloride, and extracted with 0.6M hydrochloric acid/10% sodium chloride solution and saturated sodium bicarbonate solution in this order, and the organic phases were combined, dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by column chromatography to give cationic lipid E3-1 (1.35 g). The main data of the nuclear magnetic hydrogen spectrum of E3-1 is shown below ：1H NMR(400MHz,CDCl3)δ:4.03(t,4H),3.64-3.61(m,2H),3.20-3.06(m,8H),2.91-2.59(m,6H),1.81-1.22(m,68H),0.85(t,12H). through MALDI-TOF test, and the molecular weight of E3-1 is 823.75Da.
EXAMPLE 4 cationic lipid (E4-1)
Corresponding to the general formula (1), R1、R2 in E4-1 areB1、B2 is a heptylene group, L1、L2 is an ester group (-C (=O) O-), X is N, L3 is a butylene group, R3 is a hydroxyl group, and the total molecular weight is about 852Da.
The preparation process is as follows:
Step a Compound S3-1 (2.89 g,12.0 mmol) was dissolved in dichloromethane (50 mL), then 7-bromoheptyl-N-succinimidyl carbonate (S4-1, 3.36g,10.0 mmol) and TEA (1.10 mL,15.0 mmol) were added sequentially and the reaction stirred at room temperature overnight. After the reaction is finished, the reaction solution is concentrated to obtain a crude product. Purification by column chromatography, concentration and oil pump drainage gave bromoesterified S4-2 (3.61 g).
Step b Compound S1-6 (0.18 g,2.0 mmol) was dissolved in acetonitrile (50 mL) under nitrogen, S4-2 (2.32 g,5.0 mmol) and DIPEA (0.36 g,4.0 mmol) were added sequentially with slow stirring and reacted at room temperature for about 20h. After the completion of the reaction, the reaction mixture was concentrated and dissolved in methylene chloride, and extracted with 0.6M hydrochloric acid/10% sodium chloride solution and saturated sodium bicarbonate solution in this order, and the organic phases were combined, dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by column chromatography to give cationic lipid E4-1 (1.40 g). The main data of nuclear magnetic hydrogen spectrum of E4-1 is shown below ：1H NMR(400MHz,CDCl3)δ:4.05(t,4H),3.64-3.61(m,2H),3.24-3.06(m,8H),2.90-2.61(m,6H),1.82-1.20(m,72H),0.86(t,12H). through MALDI-TOF test, and the molecular weight of E4-1 is 851.83Da.
EXAMPLE 5 cationic lipid (E5-1)
Corresponding to the general formula (1), in E5-1, R1 isR2 isB1、B2 is hexylene, L1、L2 is ester (-C (=O) O-), X is N, L3 is butylene, R3 is hydroxy, and the total molecular weight is about 823Da.
The preparation process is as follows:
Step a to a round bottom flask containing 2-hexyldecanoic acid (S5-1, 2.56g,10.0 mmol), S1-4 (2.16 g,12.0 mmol) and DMAP (0.31 g,2.5 mmol) in dichloromethane (100 mL) was added DCC (4.53 g,22.0 mmol) under nitrogen and reacted at room temperature for 16h. After the completion of the reaction, the precipitate was removed by filtration, and the filtrate was concentrated, and the obtained residue was purified by silica gel column chromatography to obtain brominated ester S5-2 (3.39 g).
Step b Compound S1-6 (0.36 g,4.0 mmol) was dissolved in acetonitrile (50 mL) under nitrogen, S5-2 (2.10 g,5.0 mmol) and DIPEA (0.36 g,4.0 mmol) were added sequentially with slow stirring and reacted at room temperature for about 20h. After the completion of the reaction, the reaction mixture was concentrated and dissolved in methylene chloride, and then extracted with 0.6M hydrochloric acid/10% sodium chloride solution and saturated sodium hydrogencarbonate solution in this order, and the organic phases were combined, dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by column chromatography to give Compound S5-3 (1.40 g).
Step c, under the protection of nitrogen, the compound S5-3 (0.86 g,2.0 mmol) is dissolved in acetonitrile (30 mL), and S5-4 (1.19 g,2.5 mmol) is added in sequence under slow stirring, wherein S5-4 is prepared from S1-4 andThe reaction was prepared by stirring the reaction at room temperature for about 20h for specific experimental procedures, see example 1.1 step b) and DIPEA (0.18 g,2.0 mmol). After the completion of the reaction, the reaction mixture was concentrated and dissolved in methylene chloride, and then extracted with 0.6M hydrochloric acid/10% sodium chloride solution and saturated sodium bicarbonate solution in this order, and the organic phases were combined, dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by column chromatography to give cationic lipid E5-1 (1.34 g). The main data of the nuclear magnetic hydrogen spectrum of E5-1 is shown below ：1H NMR(400MHz,CDCl3)δ:4.06(t,2H),4.01(t,2H),3.66-3.62(m,2H),3.22(t,2H),3.04(t,2H),3.02-2.81(m,10H),2.29-2.22(m,1H),1.92-1.21(m,68H),0.83(t,12H). through MALDI-TOF test, and the molecular weight of E5-1 is 822.77Da.
EXAMPLE 6.1 cationic lipid (E6-1)
Corresponding to the general formula (1), in E6-1, R1 isR2 isB1、B2 is hexylene, L1、L2 is ester (-C (=O) O-), X is N, L3 is butylene, R3 is hydroxy, and the total molecular weight is about 767Da.
The preparation process is as follows:
Compound S5-3 (0.86 g,2.0 mmol) was dissolved in acetonitrile (30 mL) under nitrogen, S2-2 (1.05 g,2.5 mmol) and DIPEA (0.18 g,2.0 mmol) were added sequentially with slow stirring and reacted at room temperature for about 20h with stirring. After the completion of the reaction, the reaction mixture was concentrated and dissolved in methylene chloride, and extracted with 0.6M hydrochloric acid/10% sodium chloride solution and saturated sodium bicarbonate solution in this order, and the organic phases were combined, dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by column chromatography to give cationic lipid E6-1 (1.25 g). The main data of nuclear magnetic hydrogen spectrum of E6-1 is shown below ：1H NMR(400MHz,CDCl3)δ:4.06-4.00(m,4H),3.65(t,2H),3.22-3.09(m,4H),2.90-2.75(m,6H),2.33-2.22(m,1H),1.83-1.22(m,64H),0.86(t,12H). through MALDI-TOF test, and the molecular weight of E6-1 is 766.87Da.
EXAMPLE 6.2 cationic lipid (E6-2)
Corresponding to the general formula (1), in E6-1, R1 isR2 isB1、B2 is hexylene, L1、L2 is ester (-C (=O) O-), X is N, L3 is butylene, R3 is hydroxy, and the total molecular weight is about 823Da.
The preparation process is as follows:
S6-1 is prepared by changing the raw material 2-hexyl decanoic acid in the step a into 2-octyl decanoic acid according to the method of the step a and the step b of the example 5, and then the cationic lipid E6-2 is obtained by reacting the S6-1 with the S3-2 according to the feeding amount and the operation steps of the example 6.1. The main data of nuclear magnetic hydrogen spectrum of E6-2 is shown below ：1H NMR(400MHz,CDCl3)δ:4.06-4.02(m,4H),3.65(t,2H),3.22-3.10(m,4H),2.92-2.76(m,6H),2.33-2.24(m,1H),1.79-1.22(m,72H),0.85(t,12H). through MALDI-TOF test, and the molecular weight of E6-2 is 822.62Da.
EXAMPLE 7.1 cationic lipid (E7-1)
Corresponding to the general formula (1), E7-1, R1 isR2 isB1、B2 is hexylene, L1 is an ester group (-OC (=O) O-), L2 is an ester group (-C (=O) O-), X is N, L3 is butylene, R3 is hydroxy, and the total molecular weight is about 767Da.
The preparation process is as follows:
Step a to a round bottom flask containing S7-1 (2.08 g,10.0 mmol), 7-pentadecanol (S7-2, 2.74g,12.0 mmol) and DMAP (0.31 g,2.5 mmol) in dichloromethane (100 mL) under nitrogen was added DCC (4.53 g,22.0 mmol) and reacted at room temperature for 16h. After the completion of the reaction, the precipitate was removed by filtration, and the filtrate was concentrated, and the obtained residue was purified by silica gel column chromatography to obtain a brominated ester (S7-3, 3.47 g).
Step b Compound S1-6 (0.36 g,4.0 mmol) was dissolved in acetonitrile (50 mL) under nitrogen, S7-3 (2.10 g,5.0 mmol) and DIPEA (0.36 g,4.0 mmol) were added sequentially with slow stirring and reacted at room temperature for about 20h. After the completion of the reaction, the reaction mixture was concentrated and dissolved in methylene chloride, and then extracted with 0.6M hydrochloric acid/10% sodium chloride solution and saturated sodium hydrogencarbonate solution in this order, and the organic phases were combined, dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by column chromatography to give Compound S7-4 (1.40 g).
Step c Compound S7-4 (0.86 g,2.0 mmol) was dissolved in acetonitrile (30 mL) under nitrogen, S2-2 (1.05 g,2.5 mmol) and DIPEA (0.18 g,2.0 mmol) were added sequentially with slow stirring and reacted at room temperature for about 20h with stirring. After the completion of the reaction, the reaction mixture was concentrated and dissolved in methylene chloride, and then extracted with 0.6M hydrochloric acid/10% sodium chloride solution and saturated sodium bicarbonate solution in this order, and the organic phases were combined, dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by column chromatography to give cationic lipid E7-1 (1.24 g). The main data of nuclear magnetic hydrogen spectrum of E7-1 is shown below ：1H NMR(400MHz,CDCl3)δ:4.87-4.79(m,1H),4.03-3.99(t,2H),3.64-3.58(m,2H),3.24-3.06(m,4H),2.90-2.59(m,6H),2.30-2.24(t,2H),1.81-1.22(m,64H),0.85(t,12H). through MALDI-TOF test, and the molecular weight of E7-1 is 766.71Da.
EXAMPLE 7.2 cationic lipid (E7-2)
Corresponding to the general formula (1), E7-2, R1 isR2 isB1、B2 is hexylene, L1 is an ester group (-OC (=O) O-), L2 is an ester group (-C (=O) O-), X is N, L3 is butylene, R3 is hydroxy, and the total molecular weight is about 823Da.
The preparation process is as follows:
The method of example 7, step a and step b is followed by the preparation of S7-5 by converting the starting material 7-pentadecanol in step a into 9-heptadecanol, and then the reaction of S7-5 with S3-2 is carried out according to the feed amount and the operation steps of example 7.1, thus obtaining the cationic lipid E7-2. The main data of nuclear magnetic hydrogen spectrum of E7-2 is shown below ：1H NMR(400MHz,CDCl3)δ:4.86-4.80(m,1H),4.02-3.98(t,2H),3.64-3.60(m,2H),3.26-3.08(m,4H),2.90-2.62(m,6H),2.31-2.25(t,2H),1.80-1.22(m,72H),0.85(t,12H). through MALDI-TOF test, and the molecular weight of E7-2 is 822.68Da.
Example 8 cationic lipid (E8-1)
Corresponding to the general formula (1), in E8-1, R1 isR2 isB1、B2 is hexylene, L1、L2 is ester (-C (=O) O-), X is N, L3 is butylene, R3 is hydroxy, and the total molecular weight is about 795Da.
The preparation process is as follows:
Under nitrogen, compound S5-3 (0.86 g,2.0 mmol) was dissolved in acetonitrile (30 mL), S1-5 (1.12 g,2.5 mmol) and DIPEA (0.18 g,2.0 mmol) were added sequentially under slow stirring and reacted at room temperature for about 20h. After the completion of the reaction, the reaction mixture was concentrated and dissolved in methylene chloride, and extracted with 0.6M hydrochloric acid/10% sodium chloride solution and saturated sodium bicarbonate solution in this order, and the organic phases were combined, dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by column chromatography to give cationic lipid E8-1 (1.29 g). The main data of nuclear magnetic hydrogen spectrum of E8-1 is shown below ：1H NMR(400MHz,CDCl3)δ:4.07(t,2H),4.01(t,2H),3.67-3.62(m,2H),3.22(t,2H),3.04(t,2H),2.99-2.85(m,10H),2.30-2.21(m,1H),1.93-1.47(m,20H),1.45-1.16(m,44H),0.84(t,12H). through MALDI-TOF test, and the molecular weight of E8-1 is 794.83Da.
EXAMPLE 9 cationic lipid (E9-1)
Corresponding to the general formula (1), E9-1, R1 isR2 isB1、B2 is hexylene, L1 is an ester group (-OC (=o) -), L2 is an ester group (-C (=o) O-), X is N, L3 is butylene, R3 is hydroxy, and the total molecular weight is about 795Da.
The preparation process is as follows:
Step a, under the protection of nitrogen, the compound S1-6 (0.36 g,4.0 mmol) is dissolved in acetonitrile (50 mL), and S9-1 (2.24 g,5.0 mmol) is added sequentially under slow stirring, wherein S9-1 is prepared from S6-1 andThe reaction was prepared by stirring the reaction at room temperature for about 20h for specific experimental procedures, see example 6, step b) and DIPEA (0.36 g,4.0 mmol). After the completion of the reaction, the reaction mixture was concentrated and dissolved in methylene chloride, and then extracted with 0.6M hydrochloric acid/10% sodium chloride solution and saturated sodium hydrogencarbonate solution in this order, and the organic phases were combined, dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by column chromatography to give Compound S9-2 (1.48 g).
Step b Compound S9-2 (0.91 g,2.0 mmol) was dissolved in acetonitrile (30 mL) under nitrogen, S2-2 (1.05 g,2.5 mmol) and DIPEA (0.18 g,2.0 mmol) were added sequentially with slow stirring and reacted at room temperature for about 20h with stirring. After the completion of the reaction, the reaction mixture was concentrated and dissolved in methylene chloride, and extracted with 0.6M hydrochloric acid/10% sodium chloride solution and saturated sodium bicarbonate solution in this order, and the organic phases were combined, dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by column chromatography to give cationic lipid E9-1 (1.30 g). The main data of nuclear magnetic hydrogen spectrum of E9-1 is shown below ：1H NMR(400MHz,CDCl3)δ:4.87-4.78(m,1H),4.03-3.99(t,2H),3.58(t,2H),3.24-3.06(m,4H),2.90-2.59(m,6H),2.30-2.24(t,2H),1.81-1.22(m,68H),0.85(t,12H). through MALDI-TOF test, and the molecular weight of E9-1 is 794.74Da.
EXAMPLE 10 cationic lipid (E10-1)
Corresponding to the general formula (1), R1 in E10-1 isR2 isB1、B2 is hexylene, L1 is carbonate group (-OC (=O) O-), L2 is ester group (-C (=O) O-), X is N, L3 is butylene, R3 is hydroxy, and the total molecular weight is about 783Da.
The preparation process is as follows:
Step a 6-bromohexyl-4-nitrophenyl carbonate (S10-1, 3.45g,10.0mmol, wherein S10-1 was prepared by reacting p-nitrophenyl chloroformate with 6-bromo-n-hexanol) was dissolved in dichloromethane (300 mL), S6-2 (9.12 g,40.0 mmol) was added dropwise with stirring at room temperature, followed by slow dropwise addition of pyridine (1.00 mL,12.5 mmol) over 10min, and then DMAP (0.24 g,2.0 mmol) was added in one portion. The reaction was stirred at room temperature for 16h, after the end of the reaction, extracted twice with dichloromethane, the organic phases were combined and washed with brine, then dried over anhydrous magnesium sulfate, filtered and concentrated to give the crude product. The crude product was purified by column chromatography on silica gel, and the target eluate was collected and concentrated to give S10-2 (1.21 g).
Step b Compound S1-6 (0.18 g,2.0 mmol) was dissolved in acetonitrile (50 mL) under nitrogen, S10-2 (1.06 g,2.5 mmol) and DIPEA (0.18 g,2.0 mmol) were added sequentially with slow stirring and reacted at room temperature for about 20h with stirring. After the completion of the reaction, the reaction mixture was concentrated and dissolved in methylene chloride, and then extracted with 0.6M hydrochloric acid/10% sodium chloride solution and saturated sodium bicarbonate solution in this order, and the organic phases were combined, dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by column chromatography to give Compound S10-3 (0.72 g).
Step c Compound S10-3 (0.44 g,1.0 mmol) was dissolved in acetonitrile (20 mL) under nitrogen, S2-2 (0.52 g,1.3 mmol) and DIPEA (0.09 g,1.0 mmol) were added sequentially with slow stirring and reacted at room temperature for about 20h with stirring. After the completion of the reaction, the reaction mixture was concentrated and dissolved in methylene chloride, and then extracted with 0.6M hydrochloric acid/10% sodium chloride solution and saturated sodium bicarbonate solution in this order, and the organic phases were combined, dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by column chromatography to give cationic lipid E10-1 (0.64 g). The main data of nuclear magnetic hydrogen spectrum of E10-1 is shown below ：1H NMR(400MHz,CDCl3)δ:4.71-4.68(m,1H),4.21(t,2H),4.03(t,2H),3.68-3.60(t,2H),2.55-2.46(m,10H),1.75-1.25(m,64H),0.89(t,12H). through MALDI-TOF test, and the molecular weight of E10-1 is 782.72Da.
EXAMPLE 11 cationic lipid (E11-1)
Corresponding to the general formula (1), R1 in E11-1 isR2 isB1 is pentylene, B2 is hexylene, L1 is an ester group (-OC (=o) -), L2 is an ester group (-C (=o) O-), X is N, L3 is butylene, R3 is hydroxy, and the total molecular weight is about 869Da.
The preparation process is as follows:
step a 1, 3-propanediol (S11-1, 9.50g,50 mmol) containing a TBS protected hydroxyl group was dissolved in 400mL of methylene chloride solution, pyridinium chlorochromate (PCC, 16.13g,75.0 mmol) was added, stirred at 15℃for at least 2 hours, then filtered, concentrated under reduced pressure, and purified by silica gel column chromatography to give TBS protected hydroxyl 3-hydroxypropionic acid (S11-2, 6.02 g).
Step b the above compound S11-2 (5.64 g,30.0 mmol) and 1-octanol (S11-3, 9.75g,75.0 mmol) were dissolved in 200mL of methylene chloride solution, and p-toluenesulfonic acid monohydrate (TsOH. H2 O,1.14g,6.0 mmol) and anhydrous sodium sulfate (10.65 g,75.0 mmol) were added. After stirring at 15℃for at least 24 hours, the crude product was concentrated under reduced pressure and purified by column chromatography to give TBS protected hydroxy acetal (S11-4, 2.84 g).
Step c-the above product S11-4 (2.16 g,5.0 mmol) was dissolved in THF (50 mL) and placed in a nitrogen-protected flask, tetrabutylammonium fluoride solution (TBAF, 50mL, 1M) was added and reacted overnight to remove TBS protection. Drying with anhydrous sodium sulfate, filtering, concentrating the filtrate to obtain a crude product of the compound S11-5. Purification by column chromatography, concentration and pumping-out by an oil pump gave acetal S11-5 (1.40 g, 88.6%) containing bare hydroxyl groups.
Step d to a round bottom flask containing S11-5 (0.76 g,2.4 mmol), S11-6 (0.39 g,2.0 mmol) and DMAP (61.00 mg,0.5 mmol) in dichloromethane (100 mL) was added DCC (0.91 g,4.4 mmol) under an argon atmosphere and reacted at room temperature for 16h. After the completion of the reaction, the precipitate was removed by filtration, and the filtrate was concentrated, and the obtained residue was purified by silica gel column chromatography to obtain brominated ester S11-7 (0.81 g).
Step e, under nitrogen protection, compound S1-6 (0.09 g,1.0 mmol) was dissolved in acetonitrile (20 mL), S11-7 (0.62 g,1.3 mmol) and DIPEA (0.09 g,1.0 mmol) were added sequentially with slow stirring and reacted at room temperature for about 20h with stirring. After the completion of the reaction, the reaction mixture was concentrated and dissolved in methylene chloride, and then extracted with 0.6M hydrochloric acid/10% sodium chloride solution and saturated sodium hydrogencarbonate solution in this order, and the organic phases were combined, dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by column chromatography to give Compound S11-8 (0.42 g).
Step f Compound 11-8 (0.30 g,0.6 mmol) was dissolved in acetonitrile (20 mL) under nitrogen and S1-5 (0.34 g,0.8 mmol) and DIPEA (0.05 g,0.6 mmol) were added sequentially with slow stirring and reacted at room temperature for about 20h. After the completion of the reaction, the reaction mixture was concentrated and dissolved in methylene chloride, and then extracted with 0.6M hydrochloric acid/10% sodium chloride solution and saturated sodium bicarbonate solution in this order, and the organic phases were combined, dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by column chromatography to give cationic lipid E11-1 (0.43 g). The main data of nuclear magnetic hydrogen spectrum of E11-1 is shown below ：1H NMR(400MHz,CDCl3)δ:4.64(t,1H),4.06(t,4H),3.64-3.61(m,4H),3.52-3.36(m,4H),3.02-2.81(m,10H),2.32(t,4H),1.80-1.21(m,64H),0.87(t,12H). through MALDI-TOF test, and the molecular weight of E11-1 is 868.79Da.
EXAMPLE 12 cationic lipid (E12-1)
Corresponding to the general formula (1), in E12-1, R1 isR2 isB1、B2 is heptyl, B2 is hexyl, L1 is an ester group (-OC (=o) -), L2 is an ester group (-C (=o) O-), X is N, L3 is butyl, R3 is hydroxy, and the total molecular weight is about 855Da.
The preparation process is as follows:
Step a) glycerin (S12-1, 3.09g,15.0 mmol) containing one TBS protected hydroxyl group, K2CO3 (6.21 g,45.0 mmol) and bromohexane (S12-2, 2.71g,16.5 mmol) were dissolved in 100mL of DMF under an argon atmosphere, the mixture was stirred at 110℃for 16 hours, after confirming the completion of the reaction by thin layer chromatography, the reaction solution was poured into water (100 mL) to precipitate, filtered, and further separated and purified by column chromatography to obtain the glycerin etherified product S12-3 (3.35 g, 89.3%) of TBS protected hydroxyl group.
Step b the above product S12-3 (1.88 g,5.0 mmol) was dissolved in THF (50 mL) and placed in a nitrogen-protected flask, tetrabutylammonium fluoride solution (TBAF, 50mL, 1M) was added and reacted overnight to remove TBS protection. Drying with anhydrous sodium sulfate, filtering, concentrating the filtrate to obtain a crude product of the compound S12-4. Purification by column chromatography, concentration and pumping-out by an oil pump gave a hydroxyl group-containing glycerol etherate S12-4 (1.14 g, 87.9%).
Step c to a round bottom flask containing S12-4 (0.62 g,2.4 mmol), 8-bromooctanoic acid (S12-5, 0.45g,2.0 mmol) and DMAP (61.00 mg,0.5 mmol) in dichloromethane (50 mL) was added DCC (0.91 g,4.4 mmol) under argon atmosphere and reacted at room temperature for 16h. After the completion of the reaction, the precipitate was removed by filtration, and the filtrate was concentrated, and the obtained residue was purified by silica gel column chromatography to obtain brominated ester S12-6 (0.76 g).
Step d Compound S1-6 (0.09 g,1.0 mmol) was dissolved in acetonitrile (20 mL) under nitrogen, S12-6 (0.58 g,1.3 mmol) and DIPEA (0.09 g,1.0 mmol) were added sequentially with slow stirring and reacted at room temperature for about 20h with stirring. After the completion of the reaction, the reaction mixture was concentrated and dissolved in methylene chloride, and then extracted with 0.6M hydrochloric acid/10% sodium chloride solution and saturated sodium hydrogencarbonate solution in this order, and the organic phases were combined, dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by column chromatography to give Compound S12-7 (0.39 g).
Step e Compound S12-7 (0.28 g,0.6 mmol) was dissolved in acetonitrile (20 mL) under nitrogen, S4-2 (0.37 g,0.8 mmol) and DIPEA (0.05 g,0.6 mmol) were added sequentially with slow stirring and reacted at room temperature for about 20h with stirring. After the completion of the reaction, the reaction mixture was concentrated and dissolved in methylene chloride, and then extracted with 0.6M hydrochloric acid/10% sodium chloride solution and saturated sodium bicarbonate solution in this order, and the organic phases were combined, dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by column chromatography to give cationic lipid E12-1 (0.42 g). The main data of nuclear magnetic hydrogen spectrum of E12-1 is shown below ：1HNMR(400MHz,CDCl3)δ:5.15-5.07(m,1H),4.03(t,2H),3.70(t,2H),3.58-3.50(m,4H),3.48-3.36(m,4H),3.22-2.91(m,10H),2.35-2.28(m,2H),1.96-1.47(m,20H),1.38-1.23(m,44H),0.87(t,12H). through MALDI-TOF test, and the molecular weight of E12-1 is 854.58Da.
EXAMPLE 13 cationic lipid (E13-1)
Corresponding to the general formula (1), R1 in E13-1 isR2 isB1、B2 is hexylene, L1、L2 is ester (-C (=O) O-), X is N, L3 is butylene, R3 isThe total molecular weight was about 794Da.
The preparation process is as follows:
step a 4-dimethylaminobutylamine (S13-1, 0.09g,1.0 mmol) was dissolved in acetonitrile (50 mL) under nitrogen, S5-2 (2.10 g,5.0 mmol) and DIPEA (0.36 g,4.0 mmol) were added sequentially with slow stirring and reacted at room temperature for about 20h with stirring. After the completion of the reaction, the reaction mixture was concentrated and dissolved in methylene chloride, and then extracted with 0.6M hydrochloric acid/10% sodium chloride solution and saturated sodium hydrogencarbonate solution in this order, and the organic phases were combined, dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by column chromatography to give Compound S13-2 (1.51 g).
Step b Compound S13-2 (0.91 g,2.0 mmol) was dissolved in acetonitrile (30 mL) under nitrogen, S2-2 (1.05 g,2.5 mmol) and DIPEA (0.18 g,2.0 mmol) were added sequentially with slow stirring and reacted at room temperature for about 20h with stirring. After the completion of the reaction, the reaction mixture was concentrated and dissolved in methylene chloride, and then extracted with 0.6M hydrochloric acid/10% sodium chloride solution and saturated sodium bicarbonate solution in this order, and the organic phases were combined, dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by column chromatography to give cationic lipid E13-1 (1.28 g). The main data of nuclear magnetic hydrogen spectrum of E13-1 is shown below ：1H NMR(400MHz,CDCl3)δ:4.07-4.01(m,4H),3.22-3.08(m,4H),2.92-2.76(m,8H),2.33-2.26(m,1H),2.23(s,6H),1.83-1.22(m,64H),0.87(t,12H). through MALDI-TOF test, and the molecular weight of E13-1 is 793.74Da.
EXAMPLE 14 cationic lipid (E14-1)
Corresponding to the general formula (1), in E14-1, R1 isR2 isB1、B2 is heptylene, L1 is ester group (-OC (=O) -), L2 is ester group (-C (=O) O-), X is N, L3 is butylene, and R3 isThe total molecular weight was about 882Da.
Referring to the preparation procedure of E13-1, using S13-1, S12-6 and S4-2 as raw materials, the same molar amount was used to obtain cationic lipid E14-1 (1.43 g). The main data of the nuclear magnetic hydrogen spectrum of the E14-1 is shown as follows, and the main data of the nuclear magnetic hydrogen spectrum of the E14-1 is shown as follows ：1H NMR(400MHz,CDCl3)δ:5.12-5.08(m,1H),4.04(t,2H),3.58-3.52(m,4H),3.48-3.36(m,4H),3.23-2.92(m,12H),2.32-2.28(m,2H),2.23(s,6H),1.96-1.22(m,64H),0.87(t,12H)., and the molecular weight of the E14-1 is determined to be 881.82Da through MALDI-TOF test.
EXAMPLE 15 cationic lipid (E15-1)
Corresponding to the general formula (1), E15-1, R1 isR2 isB1、B2 is hexylene, L1、L2 is ester (-C (=O) O-), X is N, L3 is butylene, R3 isThe total molecular weight was about 822Da.
Referring to the preparation procedure of E13-1, using S13-1, S5-2 and S1-5 as raw materials, the same molar amount was used to obtain cationic lipid E15-1 (1.33 g). The main data of the nuclear magnetic hydrogen spectrum of E15-1 is shown below ：4.06(t,2H),4.01(t,2H),3.63(t,2H),3.20(t,2H),3.02-2.81(m,12H),2.26(t,1H),2.20(s,6H),1.92-1.21(m,64H),0.83(t,12H). through MALDI-TOF test, and the molecular weight of E15-1 is 821.77Da.
EXAMPLE 16 cationic lipid (E16-1)
Corresponding to the general formula (1), in E16-1, R1 is undecyl and R2 isB1 is pentylene, B2 is heptylene, L1 is ester group (-OC (=O) -), L2 is ester group (-C (=O) O-), X is N, L3 is butylene, and R3 isThe total molecular weight was about 766Da.
The preparation process is as follows:
Step a S3-1 (2.89 g,12.0 mmol) was dissolved in dichloromethane (60 mL), 7-bromoheptyl-N-succinimidyl carbonate (S16-1, 3.35g,10.0 mmol) and TEA (1.10 mL,15.0 mmol) were added sequentially and the reaction stirred at room temperature overnight. After the reaction is finished, the reaction solution is concentrated to obtain a crude product. Purification by column chromatography, concentration and oil pump drainage gave bromoesterified S16-2 (3.65 g).
Step b Compound S13-1 (0.46 g,4.0 mmol) was dissolved in acetonitrile (50 mL) under nitrogen, S16-2 (2.31 g,5.0 mmol) and DIPEA (0.36 g,4.0 mmol) were added sequentially with slow stirring and reacted at room temperature for about 20h. After the completion of the reaction, the reaction mixture was concentrated and dissolved in methylene chloride, and then extracted with 0.6M hydrochloric acid/10% sodium chloride solution and saturated sodium hydrogencarbonate solution in this order, and the organic phases were combined, dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by column chromatography to give Compound S16-3 (1.62 g).
Step c-Compound S16-3 (1.00 g,2.0 mmol) was dissolved in acetonitrile (30 mL) under nitrogen, undecyl 6-bromohexanoate (S16-4, 0.87g,2.5mmol, wherein S16-4 was prepared by reacting 6-bromohexanoic acid with undecanol) and DIPEA (0.18 g,2.0 mmol) were added sequentially with slow stirring and reacted at room temperature for about 20h. After the completion of the reaction, the reaction mixture was concentrated and dissolved in methylene chloride, and then extracted with 0.6M hydrochloric acid/10% sodium chloride solution and saturated sodium bicarbonate solution in this order, and the organic phases were combined, dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by column chromatography to give cationic lipid E16-1 (1.26 g). The main data of nuclear magnetic hydrogen spectrum of E16-1 is shown below ：1H NMR(400MHz,CDCl3)δ:4.03(t,4H),3.22-3.09(m,4H),2.90-2.79(m,8H),2.30(t,2H),2.23(s,6H),1.76-1.19(m,62H),0.87(t,9H). through MALDI-TOF test, and the molecular weight of E16-1 is 765.74Da.
EXAMPLE 17 cationic lipid (E17-1)
Corresponding to the general formula (1), in E17-1, R1 isR2 isB1、B2 is hexylene, L1、L2 is ester (-C (=O) O-) and X is N, L3 is-CH2CH2OCH2CH2-,R3 is hydroxy, and the total molecular weight is about 811Da.
The preparation process is as follows:
Step a) diglycolamine (S17-1, 0.42g,4.0 mmol) was dissolved in acetonitrile (50 mL) under nitrogen, S5-2 (2.10 g,5.0 mmol) and DIPEA (0.36 g,4.0 mmol) were added in sequence with slow stirring and reacted at room temperature for about 20h with stirring. After the completion of the reaction, the reaction mixture was concentrated and dissolved in methylene chloride, and then extracted with 0.6M hydrochloric acid/10% sodium chloride solution and saturated sodium hydrogencarbonate solution in this order, and the organic phases were combined, dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by column chromatography to give Compound S16-3 (1.44 g).
Step b Compound S16-3 (0.89 g,2.0 mmol) was dissolved in acetonitrile (30 mL) under nitrogen, S1-5 (0.87 g,2.5 mmol) and DIPEA (0.18 g,2.0 mmol) were added sequentially with slow stirring and reacted at room temperature for about 20h with stirring. After the completion of the reaction, the reaction mixture was concentrated and dissolved in methylene chloride, and then extracted with 0.6M hydrochloric acid/10% sodium chloride solution and saturated sodium bicarbonate solution in this order, and the organic phases were combined, dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by column chromatography to give cationic lipid E17-1 (1.33 g). The main data of nuclear magnetic hydrogen spectrum of E17-1 is shown below ：1H NMR(400MHz,CDCl3)δ:4.06(t,2H),4.00(t,2H),3.70(t,2H),3.65-3.63(m,6H),3.20(t,2H),3.02-2.81(m,8H),2.65(t,2H),2.25(t,1H),1.80-1.19(m,60H),0.83(t,12H). through MALDI-TOF test, and the molecular weight of E17-1 is 810.74Da.
EXAMPLE 18 cationic lipid (E18-1)
Corresponding to the general formula (1), in E18-1, R1 is undecyl and R2 isB1 is pentylene, B2 butylene, L1 is an ester group (-OC (=o) -), L2 is an ester group (-C (=o) O-), X is N, L3 is-CH2CH2OCH2CH2-,R3 is hydroxy, and the total molecular weight is about 755Da.
Referring to the preparation procedure of E13-1, using S16-2, S17-1 and S16-4 as raw materials, the same molar amount was used to obtain cationic lipid E18-1 (1.24 g). The main data of nuclear magnetic hydrogen spectrum of E18-1 is shown below ：1H NMR(400MHz,CDCl3)δ:4.03(t,4H),3.71(t,2H),3.63(t,4H),3.22-2.81(m,8H),2.65(t,2H),2.30(t,2H),1.77-1.19(m,58H),0.87(t,9H). through MALDI-TOF test, and the molecular weight of E18-1 is 754.64Da.
EXAMPLE 19 cationic lipid (E19-1)
Corresponding to the general formula (1), in E19-1, R1 is undecyl and R2 isB1 is pentylene, B2 heptylene, L1 is an ester group (-OC (=o) -), L2 is an ester group (-C (=o) O-), X is N, L3 is ethylene, R3 is hydroxy, and the total molecular weight is about 711Da.
Referring to the preparation procedure of E13-1, starting from S16-2, S17-1 and S16-4, the same molar amount was used to obtain cationic lipid E19-1 (1.15 g). The main data of nuclear magnetic hydrogen spectrum of E19-1 is shown below ：1H NMR(400MHz,CDCl3)δ:4.03(t,4H),3.86-3.78(m,2H),3.22-3.09(m,4H),2.98-2.81(m,6H),2.30(t,2H),1.79-1.20(m,58H),0.88(t,9H). through MALDI-TOF test, and the molecular weight of E19-1 is 710.70Da.
EXAMPLE 20 cationic lipid (E20-1)
Corresponding to the general formula (1), in E20-1, R1 is undecyl and R2 isB1 is pentylene, B2 heptylene, L1、L2 are ester groups (-C (=O) O-), X is N, L3 is ethylene, R3 is hydroxy, and the total molecular weight is about 711Da.
The preparation process is as follows:
Compound S16-3 (0.89 g,2.0 mmol) was dissolved in acetonitrile (50 mL) under nitrogen, 5-bromopentanyl laurate (S20-1, 0.87g,2.5mmol, wherein S20-1 was prepared by reacting lauric acid with 5-bromo-1-pentanol) and DIPEA (0.18 g,2.0 mmol) were added sequentially with slow stirring, and the reaction was stirred at room temperature for about 20 hours. After the completion of the reaction, the reaction mixture was concentrated and dissolved in methylene chloride, and then extracted with 0.6M hydrochloric acid/10% sodium chloride solution and saturated sodium bicarbonate solution in this order, and the organic phases were combined, dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by column chromatography to give cationic lipid E20-1 (1.11 g). The main data of nuclear magnetic hydrogen spectrum of E20-1 is shown below ：1H NMR(400MHz,CDCl3)δ:4.03(t,4H),3.86-3.78(m,2H),3.22-3.09(m,4H),2.98-2.83(m,6H),2.32(t,2H),1.76-1.22(m,58H),0.86(t,9H). through MALDI-TOF test, and the molecular weight of E20-1 is 710.92Da.
EXAMPLE 21 cationic lipid (E21-1)
Corresponding to the general formula (1), in E21-1, R1 is undecyl and R2 isB1 is pentylene, B2 heptylene, L1 is carbonate group (-OC (=o) O-), L2 is ester group (-C (=o) O-), X is N, L3 is ethylene, R3 is hydroxy, and the total molecular weight is about 727Da.
The preparation process is as follows:
Step a S10-1 (4.14 g,12.0 mmol) was dissolved in dichloromethane (200 mL) under nitrogen, 1-undecanol (S21-1, 8.26g,48.0 mmol) was added dropwise with stirring at room temperature, followed by slow dropwise addition of pyridine (1.00 mL,15.0 mmol) over 10min, followed by one addition of DMAP (0.29 g,2.4 mmol). The reaction was stirred at room temperature for 16h, after the end of the reaction, extracted twice with dichloromethane, the organic phases were combined and washed with brine, then dried over anhydrous magnesium sulfate, filtered and concentrated to give the crude product. Purification by silica gel column separation and concentration gave 6-bromohexyl undecyl carbonate (S21-2, 1.18 g).
Step b Compound S1-7 (0.12 g,2.0 mmol) was dissolved in acetonitrile (30 mL) under nitrogen, S21-2 (1.08 g,2.5 mmol) and DIPEA (0.18 g,2.0 mmol) were added sequentially with slow stirring and reacted at room temperature for about 20h with stirring. After the completion of the reaction, the reaction mixture was concentrated and dissolved in methylene chloride, and then extracted with 0.6M hydrochloric acid/10% sodium chloride solution and saturated sodium hydrogencarbonate solution in this order, and the organic phases were combined, dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by column chromatography to give Compound S21-3 (0.60 g).
Step c Compound S21-3 (0.36 g,1.0 mmol) was dissolved in acetonitrile (20 mL) under nitrogen, S16-2 (0.58 g,1.3 mmol) and DIPEA (0.09 g,1.0 mmol) were added sequentially with slow stirring and reacted at room temperature for about 20h with stirring. After the completion of the reaction, the reaction mixture was concentrated and dissolved in methylene chloride, and then extracted with 0.6M hydrochloric acid/10% sodium chloride solution and saturated sodium bicarbonate solution in this order, and the organic phases were combined, dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by column chromatography to give cationic lipid E21-1 (0.59 g). The main data of nuclear magnetic hydrogen spectrum of E21-1 is shown below ：1HNMR(400MHz,CDCl3)δ:4.19(t,4H),4.03(t,2H),3.86-3.78(m,2H),3.22-3.09(m,4H),2.96-2.81(m,6H),1.76-1.23(m,58H),0.87(t,9H). through MALDI-TOF test, and the molecular weight of E21-1 is 726.63Da.
EXAMPLE 22 cationic lipid (E22-1)
Corresponding to the general formula (1), in E22-1, R1 is undecyl and R2 isB1 is pentylene, B2 heptylene, L1 is an ester group (-OC (=o) -), L2 is an ester group (-C (=o) O-), X is N, L3 is ethylene, R3 is hydroxy, and the total molecular weight is about 739Da.
The preparation process is as follows:
Step a, under the protection of nitrogen, the compound S1-7 (0.12 g,2.0 mmol) is dissolved in acetonitrile (30 mL), and S22-1 (1.23 g,2.5 mmol) is added in sequence under slow stirring, wherein S22-1 is prepared from 7-bromo-n-heptanol andThe reaction was prepared by referring to example 1.1, step b) and DIPEA (0.18 g,2.0 mmol) and was stirred at room temperature for about 20h. After the completion of the reaction, the reaction mixture was concentrated and dissolved in methylene chloride, and then extracted with 0.6M hydrochloric acid/10% sodium chloride solution and saturated sodium hydrogencarbonate solution in this order, and the organic phases were combined, dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by column chromatography to give Compound S22-2 (0.78 g).
Step b Compound S22-2 (0.47 g,1.0 mmol) was dissolved in acetonitrile (20 mL) under nitrogen, S16-4 (0.44 g,1.3 mmol) and DIPEA (0.09 g,1.0 mmol) were added sequentially with slow stirring and reacted at room temperature for about 20h with stirring. After the completion of the reaction, the reaction mixture was concentrated and dissolved in methylene chloride, and then extracted with 0.6M hydrochloric acid/10% sodium chloride solution and saturated sodium bicarbonate solution in this order, and the organic phases were combined, dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by column chromatography to give cationic lipid E22-1 (0.59 g). The main data of nuclear magnetic hydrogen spectrum of E22-2 is shown below ：1HNMR(400MHz,CDCl3)δ:4.03(t,4H),3.86-3.78(m,2H),3.63(t,2H),3.22-3.09(m,4H),2.99-2.81(m,6H),2.30(t,4H),1.81-1.19(m,58H),0.88(t,9H). through MALDI-TOF test, and the molecular weight of E22-1 is 738.65Da.
EXAMPLE 23 cationic lipid (E23-1)
Corresponding to the general formula (1), in E23-1, R1 is a nonylalkyl group and R2 isB1、B2 is a heptylene group, L1、L2 is an ester group (-C (=O) O-), X is N, L3 is ethylene, R3 is hydroxy, and the total molecular weight is about 739Da.
The preparation process is as follows:
Under nitrogen, compound S22-2 (0.94 g,2.0 mmol) was dissolved in acetonitrile (20 mL) and S23-1 (0.87 g,2.5 mmol) prepared by reacting 7-bromo-n-heptanol with n-decanoic acid was added sequentially with slow stirring, and the reaction was continued for about 20h at room temperature with reference to example 1.1 step b) and DIPEA (0.18 g,2.0 mmol). After the completion of the reaction, the reaction mixture was concentrated and dissolved in methylene chloride, and then extracted with 0.6M hydrochloric acid/10% sodium chloride solution and saturated sodium bicarbonate solution in this order, and the organic phases were combined, dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by column chromatography to give cationic lipid E23-1 (1.21 g). The main data of nuclear magnetic hydrogen spectrum of E23-1 is shown below ：1H NMR(400MHz,CDCl3)δ:4.03(t,4H),3.86-3.76(m,2H),3.64(t,2H),3.22-3.09(m,4H),2.98-2.81(m,6H),2.31(t,4H),1.75-1.21(m,58H),0.87(t,9H). through MALDI-TOF test, and the molecular weight of E23-1 is 738.69Da.
EXAMPLE 24 cationic lipid (E24-1)
Corresponding to the general formula (1), in E24-1, R1 is octyl and R2 isB1、B2 is a heptylene group, L1 is a carbonate group (-OC (=O) O-), L2 is an ester group (-C (=O) O-) X is N, L3 is ethylene, R3 is hydroxy, and the total molecular weight is about 741Da.
The preparation process is as follows:
Compound S22-2 (0.94 g,2.0 mmol) was dissolved in acetonitrile (20 mL) under nitrogen and S24-1 (0.88 g,2.5 mmol) prepared from 7-bromoheptyl-4-nitrophenylcarbonate and octanol was added sequentially with slow stirring, and the reaction was carried out for about 20h at room temperature with stirring for specific experimental procedures, see example 10 step a) and DIPEA (0.18 g,2.0 mmol). After the completion of the reaction, the reaction mixture was concentrated and dissolved in methylene chloride, and then extracted with 0.6M hydrochloric acid/10% sodium chloride solution and saturated sodium bicarbonate solution in this order, and the organic phases were combined, dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by column chromatography to give cationic lipid E24-1 (1.22 g). The main data of nuclear magnetic hydrogen spectrum of E24-1 is shown below ：1H NMR(400MHz,CDCl3)δ:4.19(t,4H),4.03(t,2H),3.85-3.78(m,2H),3.63(t,2H),3.22-3.09(m,4H),2.98-2.83(m,6H),2.30(t,2H),1.79-1.19(m,56H),0.87(t,9H). through MALDI-TOF test, and the molecular weight of E24-1 is 740.68Da.
EXAMPLE 25 cationic lipid (E25-1)
Corresponding to the general formula (1), R1、R2 in E25-1 areB1、B2 is hexylene, L1、L2 is ester (-C (=O) O-) and X is N, L3 isR3 is hydroxy, the total molecular weight is about 908Da.
The preparation process is as follows:
Compound 2- (4- (2-aminoethyl) piperazin-1-yl) ethanol (S25-1, 0.35g,2.0 mmol) was dissolved in acetonitrile (100 mL) under nitrogen, and S1-5 (2.24 g,5.0 mmol) and DIPE A (0.18 g,2.0 mmol) were added in sequence with slow stirring and reacted at room temperature for about 20h with stirring. After the completion of the reaction, the reaction mixture was concentrated and dissolved in methylene chloride, and extracted with 0.6M hydrochloric acid/10% sodium chloride solution and saturated sodium bicarbonate solution in this order, and the organic phases were combined, dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by column chromatography to give cationic lipid E25-1 (1.48 g). The main data of nuclear magnetic hydrogen spectrum of E25-1 is shown below ：1H NMR(400MHz,CDCl3)δ:4.03(t,4H),3.71(t,2H),3.63(t,4H),3.12-2.49(m,26H),2.31(t,4H),1.78-1.19(m,56H),0.87(t,12H). through MALDI-TOF test, and the molecular weight of E25-1 is 907.83Da.
EXAMPLE 26 cationic lipid (E26-1)
Corresponding to the general formula (1), E26-1, R1 isR2 isB1、B2 is hexylene, L1 is carbonate group (-OC (=O) O-), L2 is ester group (-C (=O) O-), X is N, and L3 isR3 is hydroxy, the total molecular weight is about 867Da.
The preparation process is as follows:
Step a Compound S25-1 (0.69 g,4.0 mmol) was dissolved in acetonitrile (50 mL) under nitrogen, S10-2 (2.17 g,5.0 mmol) and DIPEA (0.36 g,4.0 mmol) were added sequentially with slow stirring and reacted at room temperature for about 20h. After the completion of the reaction, the reaction mixture was concentrated and dissolved in methylene chloride, and then extracted with 0.6M hydrochloric acid/10% sodium chloride solution and saturated sodium hydrogencarbonate solution in this order, and the organic phases were combined, dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by column chromatography to give Compound S26-1 (1.67 g).
Step b Compound S26-1 (1.06 g,2.0 mmol) was dissolved in acetonitrile (30 mL) under nitrogen, S2-2 (1.05 g,2.5 mmol) and DIPEA (0.18 g,2.0 mmol) were added sequentially with slow stirring and reacted at room temperature for about 20h with stirring. After the completion of the reaction, the reaction mixture was concentrated and dissolved in methylene chloride, and then extracted with 0.6M hydrochloric acid/10% sodium chloride solution and saturated sodium bicarbonate solution in this order, and the organic phases were combined, dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by column chromatography to give cationic lipid E26-1 (1.39 g). The main data of nuclear magnetic hydrogen spectrum of E26-1 is shown below ：1H NMR(400MHz,CDCl3)δ:4.71-4.68(m,1H),4.21(t,2H),4.03(t,2H),3.71(t,2H),3.12-2.49(m,18H),2.55-2.46(m,4H),1.75-1.25(m,60H),0.89(t,12H). through MALDI-TOF test, and the molecular weight of E26-1 is 866.75Da.
EXAMPLE 27 cationic lipid (E27-1)
Corresponding to the general formula (1), E27-1, R1 isR2 isB1、B2 is hexylene, L1、L2 is ester (-C (=O) O-), X is N, L3 is propylene, R3 is azido, and the total molecular weight is about 778Da.
The preparation process is as follows:
Step a Compound 3-azidopropylamine (S27-1, 0.40g,4.0 mmol) was dissolved in acetonitrile (50 mL) under nitrogen, and S5-2 (2.09 g,5.0 mmol) and DIPEA (0.36 g,4.0 mmol) were added sequentially with slow stirring and reacted at room temperature for about 20h with stirring. After the completion of the reaction, the reaction mixture was concentrated and dissolved in methylene chloride, and then extracted with 0.6M hydrochloric acid/10% sodium chloride solution and saturated sodium hydrogencarbonate solution in this order, and the organic phases were combined, dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by column chromatography to give Compound S27-2 (1.41 g).
Step b Compound S27-2 (0.88 g,2.0 mmol) was dissolved in acetonitrile (30 mL) under nitrogen, S2-2 (1.05 g,2.5 mmol) and DIPEA (0.18 g,2.0 mmol) were added sequentially with slow stirring and reacted at room temperature for about 20h with stirring. After the completion of the reaction, the reaction mixture was concentrated and dissolved in methylene chloride, and then extracted with 0.6M hydrochloric acid/10% sodium chloride solution and saturated sodium bicarbonate solution in this order, and the organic phases were combined, dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by column chromatography to give cationic lipid E27-1 (1.23 g). The main data of nuclear magnetic hydrogen spectrum of E27-1 is shown below ：1H NMR(400MHz,CDCl3)δ:4.06(t,2H),4.01(t,2H),3.24-3.06(m,4H),2.90-2.59(m,6H),2.25(t,1H),1.81-1.22(m,64H),0.85(t,12H). through MALDI-TOF test, and the molecular weight of E27-1 is 777.72Da.
EXAMPLE 28 preparation of cationic Liposome nucleic acid pharmaceutical compositions
TABLE 1 formulation of liposomes and physicochemical Properties of Liposome pharmaceutical compositions
In this example, a plurality of groups of cationic liposomes were prepared for comparison, wherein the neutral lipids contained in the composition of each group of cationic liposomes were DSPC, the sterol lipids contained therein were cholesterol, and the PEGylated lipids contained therein were PEG2k-DMG (abbreviated as DMG), and only the cationic lipids were different, wherein the control group 1: cationic lipid was ALC-0315, prepared by the method disclosed in reference CN108368028A, the control group 2: cationic lipid was SM102, prepared by the method disclosed in reference CN110520409A, and the experimental group series (L-1 to L-31) of cationic lipids were the cationic lipids prepared in the examples of the present application, specifically as shown in Table 1.
Preparation of cationic Liposome nucleic acid pharmaceutical composition (LNP-mRNA) cationic lipid, DSPC, cholesterol and PEGylated lipid listed in Table 1 were dissolved in ethanol in a suitable molar ratio to obtain an ethanol phase solution, fluc-mRNA was added to 50mM citrate buffer (pH=4) in an N/P ratio of 6:1 to obtain an aqueous phase solution, the aforementioned ethanol phase solution and aqueous phase solution in a volume ratio of 1:3 were mixed and washed by multiple DPBS ultrafiltration to remove ethanol and free molecules, and finally filtered through a 0.2 μm sterile filter to obtain a cationic Liposome nucleic acid pharmaceutical composition.
EXAMPLE 29 physicochemical Property testing of cationic Liposome nucleic acid pharmaceutical compositions
Encapsulation efficiency measurement in this example, the encapsulation efficiency of the cationic liposome was measured using the Quant-it Ribogreen RNA quantitative measurement kit, and the results show that the cationic liposome of the present application has a higher encapsulation efficiency on nucleic acid drugs (mRNA), both in the range of 80% -95%, and the majority of encapsulation efficiency in the range of 85% -95%, as shown in Table 1 in particular. The results show that the entrapment rate of the cationic lipid containing multiple nitrogen branches in the application is higher or lower than that of the control group, and the entrapment rate of the lipid compound taking tertiary amine as nitrogen branches to lead out the hydrophobic fatty tail chain is lower, for example, the entrapment rates of L-1, L-2, L-3 and L-12 are lower, while the entrapment rate of the cationic lipid taking amine in the carbamate bond as nitrogen branches to lead out the hydrophobic fatty tail chain is higher, and the entrapment effect taking amine in the carbamate bond as nitrogen branches to lead out the hydrophobic fatty tail chain at one end and taking carbon branches to lead out the hydrophobic tail chain at one end is better, for example, L-8, L-9, L-10 and L-16.
Particle size measurement in this example, the particle size of LNP-mRNA was measured by Dynamic Light Scattering (DLS). The measured cationic liposome has higher size uniformity, and the PDI is less than 0.3. The cationic liposome prepared from the lipid composition of the present application has a particle size in the range of 90-120nm, as shown in Table 1.
EXAMPLE 30 biological Activity test of cationic Liposome nucleic acid pharmaceutical compositions
(1) Study of cytotoxicity (biocompatibility)
The cytotoxicity of the cationic liposome nucleic acid pharmaceutical composition of the present invention was tested by MTT staining, the cationic liposome nucleic acid pharmaceutical was dissolved in a medium to prepare the required dose, 293T cells were used as a cell model to inoculate 4×104 cells/well at a density of 100 μl/well of cell suspension into 96 well plates. After inoculation, incubation was performed in a cell incubator for 24h, and then dosing was performed at a dose of 0.2ug mRNA per well, with a corresponding volume of fresh medium added to the blank, 3 duplicate wells per group. After the composition preparation is incubated with 293T cells for 24 hours, 20 mu L of PBS buffer solution of MTT with concentration of 5mg/mL is added to each well, and after incubation with 293T cells for 4 hours, the mixed solution of the culture medium and the MTT buffer solution is sucked away, 150 mu L of DMSO is added to each well, and after shaking is completed, the absorbance is tested by an enzyme-labeled instrument. The result shows that compared with a blank control group, the cell survival rate of the cationic liposome nucleic acid pharmaceutical composition prepared by the invention is more than 95%, which indicates that the cationic liposome nucleic acid pharmaceutical composition has good biocompatibility.
(2) Investigation of mRNA transfection Rate at the cellular level
To examine the mRNA transfection efficiency at the cellular level of some cationic liposome pharmaceutical compositions (each of the groups L-CT1, L-CT2, L-1, L-8, L-9, L-10, L-11, L-12, L-16, L-22, L-23) prepared in example 28 of the present invention, a test was performed using Luciferase bioluminescence. The cationic liposome nucleic acid drug preparation was dissolved in a medium to prepare the required dose, 293T cells were used as a cell model to inoculate 4X 104 cells/well, and 100. Mu.L/well of the cell suspension was inoculated into a 96-well plate with a transparent black bottom. After inoculation, incubation in a cell incubator for 24h, followed by dosing with a dose of 0.2ug mRNA per well, the blank group was dosed with the corresponding dose of free Fluc-mRNA, each at 3 duplicate wells, after 24h of transfection, the old medium was removed, replaced with new medium containing D-sodium fluorescein (1.5 mg/mL) substrate, and after 5min incubation, bioluminescence was detected using an enzyme-labeled instrument, with stronger fluorescence indicating more Fluc-mRNA was transported into the cytoplasm and translated into the corresponding fluorescent protein. The results are shown in Table 2, wherein the relative values of fluorescence intensity are the ratio of the fluorescence intensity value of each group to the fluorescence intensity value of the blank group. The results show that compared with the blank group, the cationic liposome nucleic acid pharmaceutical composition prepared by the invention has excellent in-vitro transfection effect, and meanwhile, the transfection efficiency of most cationic liposome nucleic acid pharmaceutical compositions is higher than that of the control group, further, the tertiary amine in the cationic lipid is not as good as possible, namely ionization of more positive charges is not necessarily performed to show excellent encapsulation efficiency and transfection efficiency, the position of the ionizable tertiary amine structure is particularly important to the overall performance of the cationic lipid, and the cationic lipid of the tertiary amine in the short-chain polar heads (such as L-8, L-9, L-10, L-11, L-16 and L-23) rather than the hydrophobic long tail chains (such as L-1, L-12 and L-26) is more beneficial to the formation of the cationic liposome, can better encapsulate the nucleic acid drug, is also beneficial to the release of the nucleic acid drug from the endosome to play a role in playing a role, and therefore higher encapsulation efficiency and cell transfection efficiency are shown.
TABLE 2 relative fluorescence values for cell transfection
The foregoing description is only illustrative of the present application and is not intended to limit the scope of the application, and all equivalent structures or equivalent processes or direct or indirect application in other related arts are included in the scope of the present application. It will be apparent to those skilled in the art that the present application can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the application and without undue experimentation. While the application has been described with respect to specific embodiments, it will be appreciated that the application may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the application following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice within the art to which the application pertains.